Method for importing a disaccharide inside a cell

Abstract

Method for importing a disaccharide inside a cell The present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation. The present invention relates to methods for importing a disaccharide inside a cell wherein the disaccharide is lactose, lactulose, N-acetyllactosamine and / or lacto-N-biose. The present invention als relates to the use of the method for the production of a saccharide.

Description

[0001] Method for importing a disaccharide inside a cell

[0002] Field of the invention

[0003] The present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation. The present invention relates to methods for importing a disaccharide inside a cell wherein the disaccharide is lactose, lactulose, N-acetyllactosamine and / or lacto-N-biose. The present invention als relates to the use of the method for the production of a saccharide.

[0004] Background

[0005] Saccharides like e.g., disaccharides and oligosaccharides, are very diverse in chemical structure and are composed of miscellaneous monosaccharides, such as e.g., glucose, galactose, N-acetylglucosamine, xylose, rhamnose, fucose, mannose, N-acetylneuraminic acid, N-acetylgalactosamine, galactosamine, glucosamine, glucuronic acid, galacturonic acid. Saccharides are widely distributed in all living organisms and play important roles in a variety of physiological and pathological processes, such as cell metastasis, signal transduction, intercellular adhesion, inflammation, and immune response. Saccharides occur in free form or in a bound form, like e.g., as part of glycoproteins, glycolipids or glycosphingolipids. Economical production of these different forms of saccharides is of utmost importance to fully benefit of their biological advantages. An important group of free saccharides comprises mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (HMOs) found in mammalian and human milk, respectively. More than 150 structurally distinct human milk oligosaccharides (HMOs) have been identified to date. Although HMOs represent only a minor amount of total human milk nutrients, their beneficial effects on the development of breast-fed infants became evident over the past decades.

[0006] Description

[0007] Summary of the invention

[0008] It is an object of the present invention to provide for methods by means of which a disaccharide is imported inside a cell. Preferably said imported disaccharide is used as precursor in the production of a saccharide, preferably an oligosaccharide, more preferably a milk oligosaccharide, even more preferably a mammalian milk oligosaccharide, even more preferably a human milk oligosaccharide by said cell, in an efficient, time and cost-effective way and which yields high amounts of said saccharide. According to the invention, this and other objects are achieved by providing methods for importing said disaccharide inside a cell as described herein. The present invention also provides methods for the purification of said saccharide. Further benefits of the teachings of this invention will be apparent to one skilled in the art from reading this invention. Definitions

[0009] The words used in this specification to describe the invention and its various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus, if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.

[0010] The various aspects and embodiments of the invention disclosed herein are to be understood not only in the order and context specifically described in this specification, but to include any order and any combination thereof. Each embodiment as identified herein may be combined together unless otherwise indicated. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0011] Whenever the context requires, unless specifically stated otherwise, all words used in the singular number shall be deemed to include the plural and vice versa. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications.

[0012] In the specification, there have been disclosed embodiments of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims. It must be understood that the illustrated embodiments have been set forth only for the purposes of example and that it should not be taken as limiting the invention. It will be apparent to those skilled in the art that alterations, other embodiments, improvements, details and uses can be made consistent with the letter and spirit of the disclosure herein and within the scope of this disclosure, which is limited only by the claims, construed in accordance with the patent law, including the doctrine of equivalents. In the claims that follow, reference characters used to designate claim steps are provided for convenience of description only, and are not intended to imply any particular order for performing the steps, unless specifically stated otherwise.

[0013] Throughout the application, unless explicitly stated otherwise, the features "synthesize", "synthesized" and "synthesis" are interchangeably used with the features "produce", "produced" and "production", respectively. Throughout the application, unless explicitly stated otherwise, the expressions "capable of...<verb>" and "capable to...<verb>" are preferably replaced with the active voice of said verb and vice versa. For example, the expression "capable of expressing" is preferably replaced with "expresses" and vice versa, i.e., "expresses" is preferably replaced with "capable of expressing". In this document and in its claims, the verb "to comprise", "to have" and "to contain" and their conjugations are used in their nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Throughout the application, the verb "to comprise" may be replaced by "to consist" or "to consist essentially of" and vice versa. In addition, the verb "to consist" may be replaced by "to consist essentially of" meaning that a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In this document and in its claims, unless specifically stated otherwise, the verbs "to comprise", "to have" and "to contain", and their conjugations, may be replaced by "to consist of" (and its conjugations) or "to consist essentially of" (and its conjugations) and vice versa. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0014] Throughout the application, unless explicitly stated otherwise, the articles "a" and "an" are preferably replaced by "at least two", more preferably by "at least three", even more preferably by "at least four", even more preferably by "at least five", even more preferably by "at least six", most preferably by "at least two". The word "about" or "approximately" when used in association with a numerical value (e.g., "about 10") or with a range (e.g., "about x to approximately y") preferably means that the value or range is interpreted as being as accurate as the method used to measure it. If no error margins are specified, the expression "about" or "approximately" when used in association with a numerical value is interpreted as having the same round-off as the given value. Throughout this document and its claims, unless otherwise stated, the expression "from x to y", wherein x and y represent numerical values, refers to a range of numerical values wherein x is the lower value of the range and y is the upper value of the range. Herein, x and y are also included in the range.

[0015] According to the present invention, the term "polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triplestranded regions, or a mixture of single- and double-stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" according to the present invention. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, are to be understood to be covered by the term "polynucleotides". It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. The term "polynucleotide(s)" also embraces short polynucleotides often referred to as oligonucleotide(s).

[0016] "Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to the skilled person. The same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Furthermore, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid sidechains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation, selenoylation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.

[0017] The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions that also may contain coding and / or non-coding sequences. "Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Similarly, a "synthetic" sequence, as the term is used herein, means any sequence that has been generated synthetically and not directly isolated from a natural source. "Synthesized", as the term is used herein, means any synthetically generated sequence and not directly isolated from a natural source.

[0018] "Recombinant" means genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.

[0019] The terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically engineered" as used herein with reference to a cell or host cell are used interchangeably and indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence "foreign to said cell" or a sequence "foreign to said location or environment in said cell"). Such cells are described to be transformed with at least one heterologous or exogenous gene or are described to be transformed by the introduction of at least one heterologous or exogenous gene. Recombinant or metabolically engineered or genetically engineered or transgenic cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The terms also encompass cells that contain a nucleic acid endogenous to the cell that has been modified or its expression or activity has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, replacement of a promoter; site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis and related techniques as known to a person skilled in the art. Accordingly, a "recombinant polypeptide" is one which has been produced by a recombinant cell. The terms also encompass cells that have been modified by removing a nucleic acid endogenous to the cell by means of common well-known technologies for a skilled person (like e.g. knocking-out genes).

[0020] A "heterologous sequence" or a "heterologous nucleic acid", as used herein, is one that originates from a source foreign to the particular cell (e.g., from a different species), or, if from the same source, is modified from its original form or place in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is modified from its original form or place in the genome. The heterologous sequence may be stably introduced, e.g., by transfection, transformation, conjugation or transduction, into the genome of the host microorganism cell, wherein techniques may be applied which will depend on the cell and the sequence that is to be introduced. Various techniques are known to a person skilled in the art and are, e.g., disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The term "mutant" or "engineered" cell or microorganism as used within the context of the present invention refers to a cell or microorganism which is genetically engineered.

[0021] The term "endogenous" within the context of the present disclosure refers to any polynucleotide, polypeptide or protein sequence that is a natural part of a cell and is occurring at its natural location in the cell chromosome and of which the control of expression has not been altered compared to the natural control mechanism acting on its expression. The term "exogenous" refers to any polynucleotide, polypeptide or protein sequence which originates from outside the cell under study and not a natural part of the cell or which is not occurring at its natural location in the cell chromosome or plasmid.

[0022] The term "heterologous" when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is from a source or derived from a source other than the host organism species. In contrast a "homologous" polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to denote a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from the host organism species. When referring to a gene regulatory sequence or to an auxiliary nucleic acid sequence used for maintaining or manipulating a gene sequence (e.g. a promoter, a 5' untranslated region, 3' untranslated region, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site, etc.), "heterologous" means that the regulatory sequence or auxiliary sequence is not naturally associated with the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome. Thus, a promoter operably linked to a gene to which it is not operably linked to in its natural state (i.e. in the genome of a non- genetically engineered organism) is referred to herein as a "heterologous promoter," even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.

[0023] The term "modified expression" of a gene relates to a change in expression compared to the wild-type expression of said gene in any phase of the production process of the desired non-charged (neutral) oligosaccharide as described herein. Said modified expression is either a lower or higher expression compared to the wild-type, wherein the term "higher expression" is also defined as "overexpression" of said gene in the case of an endogenous gene or "expression" in the case of a heterologous gene that is not present in the wild-type strain. Lower expression is obtained by means of common well-known technologies for a skilled person (such as the usage of siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc.) which are used to change the genes in such a way that they are "less-able" (i.e., statistically significantly 'less-able' compared to a functional wild-type gene) or completely unable (such as knocked-out genes) to produce functional final products. The term "riboswitch" as used herein is defined to be part of the messenger RNA that folds into intricate structures that block expression by interfering with translation. Binding of an effector molecule induces conformational change(s) permitting regulated expression post-transcriptionally. Next to changing the gene of interest in such a way that lower expression is obtained as described above, lower expression can also be obtained by changing the transcription unit, the promoter, an untranslated region, the ribosome binding site, the Shine Dalgarno sequence or the transcription terminator. Lower expression or reduced expression can for instance be obtained by mutating one or more base pairs in the promoter sequence or changing the promoter sequence fully to a constitutive promoter with a lower expression strength compared to the wild-type or an inducible promoter which result in regulated expression or a repressible promoter which results in regulated expression. Overexpression or expression is obtained by means of common well-known technologies for a skilled person (such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids), wherein said gene is part of an "expression cassette" that relates to any sequence in which a promoter sequence, untranslated region sequence (containing either a ribosome binding sequence, Shine Dalgarno or Kozak sequence), a coding sequence and optionally a transcription terminator is present, and leading to the expression of a functional active protein. Said expression is either constitutive or conditional or regulated or tuneable.

[0024] The term "constitutive expression" is defined as expression that is not regulated by transcription factors other than the subunits of RNA polymerase (e.g., the bacterial sigma factors like s70, s54, or related s- factors and the yeast mitochondrial RNA polymerase specificity factor MTFl that co-associate with the RNA polymerase core enzyme) under certain growth conditions. Non-limiting examples of such transcription factors are CRP, Lacl, ArcA, Cra, IcIR in E. coli, or, Aft2p, Crzlp, Skn7 in Saccharomyces cerevisiae, or, DeoR, GntR, Fur in B. subtilis. These transcription factors bind on a specific sequence and may block or enhance expression in certain growth conditions. The RNA polymerase is the catalytic machinery for the synthesis of RNA from a DNA template. RNA polymerase binds a specific DNA sequence to initiate transcription, for instance via a sigma factor in prokaryotic hosts or via MTFl in yeasts. Constitutive expression offers a constant level of expression with no need for induction or repression.

[0025] The term "regulated expression" is defined as expression that is regulated by transcription factors other than the subunits of RNA polymerase (e.g. bacterial sigma factors) under certain growth conditions. Examples of such transcription factors are described above. Commonly expression regulation is obtained by means of an inducer, such as but not limited to IPTG, arabinose, rhamnose, fucose, allo-lactose or pH shifts, or temperature shifts or carbon depletion or substrates or the produced product.

[0026] The term "control sequences" refers to sequences recognized by the cells transcriptional and translational systems, allowing transcription and translation of a polynucleotide sequence to a polypeptide. Such DNA sequences are thus necessary for the expression of an operably linked coding sequence in a particular host cell, cell or organism. Such control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, transcription terminator sequences. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. DNA for a presequence or secretory leader may be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Said control sequences can furthermore be controlled with external chemicals, such as, but not limited to, IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of said polynucleotide to a polypeptide.

[0027] Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. The term "wildtype" refers to the commonly known genetic or phenotypical situation as it occurs in nature.

[0028] The term "modified expression of a protein" as used herein refers to i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein, iii) expression and / or overexpression of a variant protein that has a higher activity compared to the wild-type (i.e. native in the expression host) protein, iv) reduced expression of an endogenous protein or v) expression and / or overexpression of a variant protein that has a reduced activity compared to the wild-type (i.e. native in the expression host) protein. Preferably, the term "modified expression of a protein" as used herein refers to i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein or iii) expression and / or overexpression of a variant protein that has a higher activity compared to the wild-type (i.e. native in the expression host) protein.

[0029] The term "modified activity" of a protein relates to a non-native activity of the protein in any phase of the production process of the desired saccharide as described herein. The term "non-native", as used herein with reference to the activity of a protein indicates that the protein has been modified to have an abolished, impaired, reduced, delayed, higher, accelerated or improved activity compared to the native activity of said protein. A modified activity of a protein is obtained by modified expression of said protein or is obtained by expression of a modified, i.e., mutant form of the protein. A mutant form of the protein can be obtained by expression of a mutant form of the gene encoding the protein, e.g., comprising a deletion, an insertion and / or a mutation of one or more nucleotides compared to the native gene sequence. A mutant form of a gene can be obtained by techniques well-known to a person skilled in the art, such as but not limited to site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis. The term "non-native", as used herein with reference to a cell producing a saccharide as described herein, indicates that said saccharide is i) not naturally produced or ii) when naturally produced not in the same amounts by the cell; and that the cell has been genetically engineered to be able to produce said saccharide or to have a higher production of the saccharide.

[0030] As used herein, the term "mammary cell(s)" generally refers to mammalian mammary epithelial cell(s), mammalian mammary-epithelial luminal cell(s), or mammalian epithelial alveolar cell(s), or any combination thereof. As used herein, the term "mammary-like cell(s)" generally refers to mammalian cell(s) having a phenotype / genotype similar (or substantially similar) to natural mammalian mammary cell(s) but is / are derived from mammalian non-mammary cell source(s). Such mammalian mammary-like cell (s) may be engineered to remove at least one undesired genetic component and / or to include at least one predetermined genetic construct that is typical of a mammalian mammary cell. Non-limiting examples of mammalian mammary-like cell(s) may include mammalian mammary epithelial-like cell(s), mammalian mammary epithelial luminal-like cell(s), mammalian non-mammary cell(s) that exhibits one or more characteristics of a cell of a mammalian mammary cell lineage, or any combination thereof. Further nonlimiting examples of mammalian mammary-like cell(s) may include mammalian cell(s) having a phenotype similar (or substantially similar) to natural mammalian mammary cell (s), or more particularly a phenotype similar (or substantially similar) to natural mammalian mammary epithelial cell(s). A mammalian cell with a phenotype or that exhibits at least one characteristic similar to (or substantially similar to) a natural mammalian mammary cell or a mammalian mammary epithelial cell may comprise a mammalian cell (e.g., derived from a mammary cell lineage or a non-mammary cell lineage) that exhibits either naturally, or has been engineered to, be capable of expressing at least one milk component.

[0031] As used herein, the term "non-mammary cell(s)" may generally include any mammalian cell of non- mammary lineage. In the context of the invention, a non-mammary cell can be any mammalian cell capable of being engineered to express at least one milk component. Non-limiting examples of such non- mammary cell(s) include hepatocyte(s), blood cell(s), kidney cell(s), cord blood cell(s), epithelial cell(s), epidermal cell(s), myocyte(s), fibroblast(s), mesenchymal cell(s), or any combination thereof. In some instances, molecular biology and genome editing techniques can be engineered to eliminate, silence, or attenuate myriad genes simultaneously.

[0032] "Variant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to the persons skilled in the art.

[0033] In some embodiments, the present invention contemplates making functional variants by modifying the structure of a polypeptide, like e.g. a membrane protein, as used in the present invention. Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof. For instance, a variant can be produced as a fusion protein comprising at least one portion of a polypeptide of present invention fused to at least one portion comprising a peptide tag. Said peptide tag may be used to assist protein folding of said polypeptide, assist post expression purification, protect the polypeptide from the action of degradative enzymes, and / or assist the polypeptide in passing through the cell membrane. Examples of said peptide tag comprise, e.g., a SUMO tag, an MBP tag, a His tag, a FLAG tag, a Strep-ll tag, a Halo-tag, a NusA tag, thioredoxin, GST and / or a Fh8-tag. The fusion protein may be designed to include at least one cleavable peptide linker so that the polypeptide of interest can be subsequently recovered from the fusion protein. The fusion protein may be designed to include a plurality of inclusion body tags, cleavable peptide linkers, and regions encoding the polypeptide of interest.

[0034] In case of amino acid substitution, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of a polypeptide of the invention results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type polypeptide.

[0035] "Fragment", with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule, particularly a part of a polynucleotide that retains a usable, functional characteristic of the full-length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A "polynucleotide fragment" refers to any subsequence of a polynucleotide SEQ ID NO, typically, comprising or consisting of at least about 9, 10, 11, 12 consecutive nucleotides from said polynucleotide SEQ ID NO, for example at least about 30 nucleotides or at least about 50 nucleotides of any of the polynucleotide sequences provided herein. Exemplary fragments can additionally or alternatively include fragments that comprise, consist essentially of, or consist of a region that encodes a conserved family domain of a polypeptide. Exemplary fragments can additionally or alternatively include fragments that comprise a conserved domain of a polypeptide. As such, a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of said polynucleotide SEQ ID NO wherein no more than about 200, 150, 100, 50 or 25 consecutive nucleotides are missing, preferably no more than about 50 consecutive nucleotides are missing, and which retains a usable, functional characteristic (e.g. activity) of the full-length polynucleotide molecule which can be assessed by the skilled person through routine experimentation. Alternatively, a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of an amount of consecutive nucleotides from said polynucleotide SEQ ID NO and wherein said amount of consecutive nucleotides is at least 50.0 %, 60.0 %, 70.0 %, 80.0 %, 81.0 %, 82.0 %, 83.0 %, 84.0 %, 85.0 %, 86.0 %, 87.0 %, 88.0 %, 89.0 %, 90.0 %, 91.0 %, 92.0 %, 93.0 %, 94.0 %, 95.0 %, 95.5%, 96.0 %, 96.5 %, 97.0 %, 97.5 %, 98.0 %, 98.5 %, 99.0 %, 99.5 %, 100 %, preferably at least 80.0 %, more preferably at least 85.0 %, even more preferably at least 87.0 %, even more preferably at least 90.0 %, even more preferably at least 95.0 %, most preferably at least 97.0 %, of the full-length of said polynucleotide SEQ ID NO and retains a usable, functional characteristic (e.g. activity) of the full-length polynucleotide molecule which can be routinely assessed by the skilled person. As such, a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of said polynucleotide SEQ ID NO, wherein an amount of consecutive nucleotides is missing and wherein said amount is no more than 50.0 %, 40.0 %, 30.0 % of the full-length of said polynucleotide SEQ ID NO, preferably no more than 20.0 %, 15.0 %, 10.0 %, 9.0 %, 8.0 %, 7.0 %, 6.0 %, 5.0 %, 4.5 %, 4.0 %, 3.5 %, 3.0 %, 2.5 %, 2.0 %, 1.5 %, 1.0 %, 0.5 %, more preferably no more than 15.0 %, even more preferably no more than 10.0 %, even more preferably no more than 5.0 %, most preferably no more than 2.5 %, of the full-length of said polynucleotide SEQ ID NO and wherein said fragment retains a usable, functional characteristic (e.g. activity) of the full-length polynucleotide molecule which can be routinely assessed by the skilled person.

[0036] "Fragment", with respect to a polypeptide, refers to a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. A "subsequence of the polypeptide" or "a stretch of amino acid residues" as described herein refers to a sequence of contiguous amino acid residues derived from the polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, for example at least about 10 amino acid residues in length, for example at least about 20 amino acid residues in length, for example at least about 30 amino acid residues in length, for example at least about 100 amino acid residues in length, for example at least about 150 amino acid residues in length, for example at least about 200 amino acid residues in length. As such, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably means a polypeptide sequence which comprises or consists of said polypeptide SEQ ID NO (or UniProt ID) wherein no more than about 200, 150, 125, 100, 80, 60, 50, 40, 30, 20 or 15 consecutive amino acid residues are missing, preferably no more than about 100 consecutive amino acid residues are missing, more preferably no more than about 50 consecutive amino acid residues are missing, even more preferably no more than about 40 consecutive amino acid residues are missing, and performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide which can be routinely assessed by the skilled person. Alternatively, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably means a polypeptide sequence which comprises or consists of an amount of consecutive amino acid residues from said polypeptide SEQ ID NO (or UniProt ID) and wherein said amount of consecutive amino acid residues is at least 50.0 %, 60.0 %, 70.0 %, 80.0 %, 81.0 %, 82.0 %, 83.0 %, 84.0 %, 85.0 %, 86.0 %, 87.0 %, 88.0 %, 89.0 %, 90.0 %, 91.0 %, 92.0 %, 93.0 %, 94.0 %, 95.0 %, 95.5%, 96.0 %, 96.5 %, 97.0 %, 97.5 %, 98.0 %, 98.5 %, 99.0 %, 99.5 %, 100 %, preferably at least 80.0 %, more preferably at least 85.0 %, even more preferably at least 87.0%, even more preferably at least 90.0 %, even more preferably at least 95.0 %, most preferably at least 97.0 % of the full-length of said polypeptide SEQ ID NO (or UniProt ID) and which performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide which can be routinely assessed by the skilled person. As such, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably means a polypeptide sequence which comprises or consists of said polypeptide SEQ ID NO (or UniProt ID), wherein an amount of consecutive amino acid residues is missing and wherein said amount is no more than 50.0 %, 40.0 %, 30.0 % of the full-length of said polypeptide SEQ ID NO (or UniProt ID), preferably no more than 20.0 %, 15.0 %, 10.0 %, 9.0 %, 8.0 %, 7.0 %, 6.0 %, 5.0 %, 4.5 %, 4.0 %, 3.5 %, 3.0 %, 2.5 %, 2.0 %, 1.5 %, 1.0 %, 0.5 %, more preferably no more than 15.0 %, even more preferably no more than 10.0 %, even more preferably no more than 5.0 %, most preferably no more than 2.5 %, of the full-length of said polypeptide SEQ ID NO (or UniProt ID) and which performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide which can be routinely assessed by the skilled person.

[0037] Throughout the application, the sequence of a polypeptide can be represented by a SEQ ID NO or alternatively by an UniProt ID. Therefore, the terms "polypeptide SEQ ID NO" and "polypeptide UniProt ID" can be interchangeably used, unless explicitly stated otherwise.

[0038] A "functional fragment" of a polypeptide has at least one property or activity of the polypeptide from which it is derived, preferably to a similar or greater extent. A functional fragment can, for example, include a functional domain or conserved domain of a polypeptide. It is understood that a polypeptide or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the polypeptide's activity. By conservative substitutions is intended substitutions of one hydrophobic amino acid for another or substitution of one polar amino acid for another or substitution of one acidic amino acid for another or substitution of one basic amino acid for another etc. Preferably, by conservative substitutions is intended combinations such as glycine by alanine and vice versa; valine, isoleucine and leucine by methionine and vice versa; aspartate by glutamate and vice versa; asparagine by glutamine and vice versa; serine by threonine and vice versa; lysine by arginine and vice versa; cysteine by methionine and vice versa; and phenylalanine and tyrosine by tryptophan and vice versa.

[0039] Homologous sequences as used herein describes those nucleotide sequences that have sequence similarity and encode polypeptides that share at least one functional characteristic such as a biochemical activity. More specifically, the term "functional homolog" as used herein describes those polypeptides that have sequence similarity (in other words, homology) and at the same time have at least one functional similarity such as a biochemical activity (Altenhoff et al., PLoS Comput. Biol. 8 (2012) el002514). Homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of the nucleotides or polypeptides of interest. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using the amino acid sequence of a reference polypeptide sequence. The amino acid sequence is, in some instances, deduced from the nucleotide sequence. Typically, those polypeptides in the database that have greater than 40 % sequence identity to a polypeptide of interest are candidates for further evaluation for suitability as a homologous polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another or substitution of one acidic amino acid for another or substitution of one basic amino acid for another etc. Preferably, by conservative substitutions is intended combinations such as glycine by alanine and vice versa; valine, isoleucine and leucine by methionine and vice versa; aspartate by glutamate and vice versa; asparagine by glutamine and vice versa; serine by threonine and vice versa; lysine by arginine and vice versa; cysteine by methionine and vice versa; and phenylalanine and tyrosine by tryptophan and vice versa. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated.

[0040] A domain can be characterized, for example, by a Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427- D432), an IPR (InterPro domain) (http: / / ebi.ac.uk / interpro) (Mitchell et al., Nucleic Acids Res. 47 (2019) D351-D360), a Conserved Domain Database (CDD) designation (https: / / www.ncbi.nlm.nih.gov / cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268) and / or a PTHR domain - also known as a PANTHER domain - (http: / / www.pantherdb.org) (Mi et al., Nucleic Acids. Res. 41 (2013) D377-D386. InterPro provides functional analysis of proteins by classifying them into families and predicting domains and important sites. To classify proteins in this way, InterPro uses predictive models, known as signatures, provided by several different databases (referred to as member databases) that make up the InterPro consortium. Protein signatures from these member databases are combined into a single searchable resource, capitalizing on their individual strengths to produce a powerful integrated database and diagnostic tool. It should be understood for those skilled in the art that for the databases used herein, comprising Pfam 32.0 (released Sept 2018), CDD v3.20 (released 06thOctober 2022), InterPro 98.0 (released 25thJanuary 2024) and PANTHER 18.0 (released 17thSeptember 2023), the content of each database is fixed at each release and is not to be changed. When the content of a specific database is changed, this specific database receives a new release version with a new release date. All release versions for each database with their corresponding release dates and specific content as annotated at these specific release dates are available and known to those skilled in the art. Protein or polypeptide sequence information and functional information can be provided by a comprehensive resource for protein sequence and annotation data like e.g., the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489). UniProt comprises the expertly and richly curated protein database called the UniProt Knowledgebase (UniProtKB), together with the UniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc). The UniProt identifiers (UniProt ID) are unique for each protein present in the database. Throughout the application, the sequence of a polypeptide is represented by a SEQ ID NO or an UniProt ID. Unless stated otherwise, the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database (www.uniprot.org) version release 2021_03 and consulted on 09 June 2021.

[0041] The terms "identical" or "percent identity" or "% identity" in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection. For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are inputted into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the % sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. The percentage of sequence identity can be, preferably is, determined by alignment of the two sequences and identification of the number of positions with identical residues divided by the number of residues in the shorter of the sequences x 100. Percent identity may be calculated globally over the full-length sequence of a given SEQ ID NO, i.e. the reference sequence, resulting in a global % identity score. Alternatively, % identity may be calculated over a partial sequence of the reference sequence, resulting in a local percent identity score. A partial sequence preferably means at least about 50 %, 60 %, 70 %, 80 %, 90 % or 95 % of the full-length reference sequence. In another preferred embodiment, a partial sequence of a reference polypeptide sequence means a stretch of at least 150 amino acid residues up to the total number of amino acid residues of a reference polypeptide sequence. In another more preferred embodiment, a partial sequence of a reference polypeptide sequence means a stretch of at least 200 amino acid residues up to the total number of amino acid residues of a reference polypeptide sequence. Using the full-length of the reference sequence in a local sequence alignment results in a global percent identity score between the test and the reference sequence. Percent identity can be determined using different algorithms like for example BLAST and PSI-BLAST (Altschul et al., 1997, Nucleic Acids Res 25: 17, 3389-402), the Clustal Omega method (Sievers et al., 2011, Mol. Syst. Biol. 7:539), the MatGAT method (Campanella et al., 2003, BMC Bioinformatics, 4:29) or EMBOSS Needle.

[0042] As used herein, a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence having 40 % or more sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the amino acid sequence has 40 %, 41 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %,

[0043] 48 %, 49 %, 50 %, 51 %, 52 %, 53 %, 54 %, 55 %, 56 %, 57 %, 58 %, 59 %, 60 %, 61 %, 62 %, 63 %, 64 %, 65

[0044] %, 66 %, 67 %, 68 %, 69 %, 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %,

[0045] 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 90.5 %, 91 %, 91.5 %, 92 %, 92.5 %, 93 %, 93.5 %, 94 %,

[0046] 94.5 %, 95 %, 95.5 %, 96 %, 96.5 %, 97 %, 97.5 %, 98 %, 98.5 %, 99 %, 99.5 %, 99.6 %, 99.7 %, 99.8 %, 99.9 %, 100 % sequence identity to the full-length of the amino acid sequence of the reference polypeptide sequence.

[0047] Throughout the application, unless explicitly specified otherwise, a polypeptide comprising, consisting of or having an amino acid sequence having 40 % or more sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ ID NO or UniProt ID, preferably has 40 %, 41 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, 49 %, 50 %, 51 %, 52 %, 53 %, 54 %, 55 %, 56 %, 57 %,

[0048] 58 %, 59 %, 60 %, 61 %, 62 %, 63 %, 64 %, 65 %, 66 %, 67 %, 68 %, 69 %, 70 %, 71 %, 72 %, 73 %, 74 %, 75

[0049] %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %,

[0050] 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 %, more preferably has at least 50 %, even more preferably has at least 55 %, even more preferably has at least 60 %, even more preferably has at least 65 %, even more preferably has at least 70 %, even more preferably has at least 75 %, even more preferably has at least 80 %, even more preferably has at least 90 %, even more preferably has at least 95 %, even more preferably has at least 97.5 %, sequence identity to the full length reference sequence.

[0051] For the purposes of this invention, percent identity is determined using MatGAT2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29). The following default parameters for protein are employed: (1) Gap cost Existence: 12 and Extension: 2; (2) The Matrix employed was BLOSUM50. In a preferred embodiment, sequence identity is calculated based on the full-length sequence of a given SEQ ID NO, i.e. the reference sequence, or a part thereof. Part thereof preferably means at least 50 %, 60 %, 70 %, 80 %, 90 % or 95 % of the complete reference sequence.

[0052] The terms "sialic acid", "N-acetylneuraminate", "N-acylneuraminate", "N-acetylneuraminic acid" are used interchangeably and refer to an acidic sugar comprising but not limited to Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO).

[0053] The term "glycosyltransferase" as used herein refers to an enzyme capable to catalyse the transfer of a sugar moiety of a donor to a specific acceptor, forming glycosidic bonds. Said donor can be a precursor as defined herein. A classification of glycosyltransferases using nucleotide diphospho-sugar, nucleotide monophospho-sugar and sugar phosphates and related proteins into distinct sequence-based families has been described (Campbell et al., Biochem. J. 326, 929-939 (1997)) and is available on the CAZy (CArbohydrate-Active EnZymes) website (www.cazy.org). As used herein the glycosyltransferase can be selected from the list comprising but not limited to: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N- acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino- 4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.

[0054] The term "saccharide" as used herein refers to a sugar selected from the list comprising, consisting of or consisting essentially of monosaccharide, phosphorylated monosaccharide, activated monosaccharide, disaccharide, oligosaccharide and polysaccharide.

[0055] The term "monosaccharide" as used herein refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed either an aldose or ketose, and contains one or more hydroxyl groups per molecule. Monosaccharides are saccharides containing only one simple sugar.

[0056] The term "phosphorylated monosaccharide" as used herein refers to a monosaccharide that is phosphorylated. Examples of phosphorylated monosaccharides include but are not limited to glucose-1- phosphate, glucose-6-phosphate, glucose-l,6-bisphosphate, galactose-l-phosphate, fructose-6- phosphate, fructose-l,6-bisphosphate, fructose-l-phosphate, glucosamine-l-phosphate, glucosamine-6- phosphate, N-acetylglucosamine-l-phosphate, mannose-l-phosphate, mannose-6-phosphate or fucose- 1-phosphate. Some, but not all, of these phosphorylated monosaccharides are precursors or intermediates for the production of activated monosaccharide.

[0057] The terms "activated monosaccharide", "nucleotide-activated sugar", "nucleotide-sugar", "activated sugar", "nucleoside" or "nucleotide donor" are used herein interchangeably and refer to activated forms of monosaccharides. Examples of activated monosaccharides include but are not limited to UDP-N- acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2- acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2- acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2- acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L- QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose, CMP-sialic acid, CMP-2-keto-3- deoxymanno-octulonic acid (CMP-KDO), GDP-fucose (GDP-Fuc), GDP-rhamnose and UDP-xylose. Nucleotide-sugars act as glycosyl donors in glycosylation reactions. Glycosylation reactions are reactions that are catalysed by glycosyltransferases. The term "CMP-sialic acid" as used herein refers to a nucleotide-activated form of sialic acid comprising but not limited to CMP-Neu5Ac, CMP-Neu4Ac, CMP- Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N- glycolylneuraminic acid (CMP-Neu5Gc) and CMP-KDO.

[0058] The term "disaccharide" as used herein refers to a saccharide polymer containing two simple sugars, i.e. monosaccharides. Examples of disaccharides comprise lactose (Gal-pi,4-Glc), lactulose (Gal-pi,4-Fruc), lacto-N-biose (Gal-pi,3-GlcNAc), N-acetyllactosamine (Gal-pi,4-GlcNAc), LacDiNAc (GalNAc-pi,4- GIcNAc), N-acetylgalactosaminylglucose (GalNAc-pi,4-Glc), Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal, fucopyranosyl- (l-4)-N-glycolylneuraminic acid (Fuc-(l-4)-Neu5Gc), sucrose (Glc-al,2-Fru), maltose (Glc- al,4-Glc) and melibiose (Gal-al,6-Glc).

[0059] The term "disaccharide having a Gal residue at its non-reducing end" as used herein refers to a saccharide polymer containing two monosaccharides, of which one is galactose wherein said galactose is positioned at the non-reducing end of said disaccharide. Examples of disaccharides having a Gal residue at their nonreducing end comprise lactose (Gal-pi,4-Glc), lactulose (Gal-pi,4-Fruc), lacto-N-biose (LNB, Gal-pi,3- GIcNAc), N-acetyllactosamine (LacNAc, Gal-pi,4-GlcNAc) and melibiose (Gal-al,6-Glc).

[0060] "Oligosaccharide" as the term is used herein and as generally understood in the state of the art, refers to a saccharide polymer containing a small number, typically three to twenty, preferably three to ten, of simple sugars, i.e., monosaccharides. The oligosaccharide as used in the present invention can be a linear structure or can include branches. The linkage (e.g., glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.) between two sugar units can be expressed, for example, as 1,4, l->4, or (1-4), used interchangeably herein. For example, the terms "Gal-bl,4-Glc", "Gal-pi,4-Glc", "b-Gal-(l->4)-Glc", "P-Gal- (l->4)-Glc", "Galbetal-4-Glc", "Gal-b(l-4)-Glc" and "Gal-P(l-4)-Glc" have the same meaning, i.e. a beta- glycosidic bond links carbon-1 of galactose (Gal) with the carbon-4 of glucose (Glc). Each monosaccharide can be in the cyclic form (e.g., pyranose or furanose form). Linkages between the individual monosaccharide units may include alpha l->2, alpha l->3, alpha l->4, alpha l->6, alpha 2->l, alpha 2->3, alpha 2->4, alpha 2->6, beta l->2, beta l->3, beta l->4, beta l->6, beta 2->l, beta 2->3, beta 2->4, and beta 2->6. An oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only alpha- glycosidic or only beta-glycosidic bonds. The term "polysaccharide" refers to a compound consisting of a large number, typically more than twenty, of monosaccharides linked glycosidically.

[0061] Examples of oligosaccharides include but are not limited to Lewis-type antigen oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigen, enterobacterial common antigen (ECA), the glycan chain present in lipopolysaccharides (LPS), the oligosaccharide repeats present in capsular polysaccharides, peptidoglycan (PG), amino-sugars, antigens of the human ABO blood group system, noncharged (neutral) oligosaccharides, negatively charged oligosaccharides, fucosylated oligosaccharides, sialylated oligosaccharides, N-acetylglucosamine containing oligosaccharides, N-acetyllactosamine containing oligosaccharides, lacto-N-biose containing oligosaccharides, lactose containing oligosaccharides, non-fucosylated non-charged (neutral) oligosaccharides, N-acetyllactosamine containing fucosylated oligosaccharides, N-acetyllactosamine non-fucosylated oligosaccharides, lacto-N- biose containing fucosylated oligosaccharides, lacto-N-biose containing non-fucosylated oligosaccharides, N-acetyllactosamine containing negatively charged oligosaccharides, lacto-N-biose containing negatively charged oligosaccharides, animal oligosaccharides, preferably selected from the group consisting of N- glycans and O-glycans, and plant oligosaccharides, preferably selected from the group consisting of N- glycans and O-glycans.

[0062] "Charged oligosaccharides" are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits including N-acetylneuraminic acid (Neu5Ac), commonly known as sialic acid, N- glycolylneuraminic acid (Neu5Gc), glucuronate and galacturonate. Charged oligosaccharides are also referred to as acidic oligosaccharides. Sialic acid belongs to the family of derivatives of neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid). Neu5Gc is a derivative of sialic acid, which is formed by hydroxylation of the N-acetyl group at C5 of Neu5Ac. The terms "sialic acid", "N- acetylneuraminate", "N-acylneuraminate", "N-acetylneuraminic acid" and "Neu(n)Ac molecule" are used interchangeably and refer to an acidic sugar with a nine-carbon backbone comprising but not limited to Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and KDO (2-keto-3-deoxymanno- octulonic acid). The terms "negatively charged oligosaccharide" or "acidic oligosaccharide" are used interchangeably and refer to an oligosaccharide with a negative charge. In a preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide. As used herein, a 'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide, i.e., an oligosaccharide having one or more sialic acid residue(s). It has an acidic nature. Some examples are 3'SL (3'-sialyllactose, Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 6'SL (6'sialyllactose, Neu5Ac-a2,6-Gal- pi,4-Glc), 8'SL (8'sialyllactose, Neu5Ac-a2,8-Gal-pi,4-Glc), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac- a2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-pi,4-Glc), 6'-sialyllactosamine, oligosaccharides comprising 6'sialyllactose, SGG hexasaccharide (Neu5Aca-2,3Gaip -l,3GalNac -l,3Gala-l,4Gaip-l,4Gal), sialylated tetrasaccharide, sialylated pentasaccharide, sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialyllacto-N-neotetraose, LSTc (Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), LSTd (Neu5Ac-a2,3- Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a (LSTa, Neu5Ac-a2,3-Gal-pi,3- GlcNAc-pi,3-Gal-pi,4-Glc), disialyllacto-N-hexaose I, sialyllacto-N-tetraose b (LSTb, Gal-pi,3-(Neu5Ac- a2,6)-GlcNAc-pi,3-Gal-pi,4-Glc), 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyl lacto-N- fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose and oligosaccharides bearing one or several sialic acid residue(s).

[0063] The terms 'neutral oligosaccharide' and 'non-charged oligosaccharide' as used herein are used interchangeably and refer, as generally understood in the state of the art, to an oligosaccharide that has no negative charge originating from a carboxylic acid group. Neutral oligosaccharides are non-sialylated oligosaccharides, and thus do not contain an acidic monosaccharide subunit. Neutral oligosaccharides comprise non-charged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure as well as non-charged non-fucosylated oligosaccharides that lack any fucose subunit. Examples of such neutral oligosaccharides comprise 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4- fucosyllactose (4FL), 6-fucosyllactose (6FL), 2', 3-difucosyllactose (diFL), lacto-N-triose II (LN3), lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI, lacto-N-neofucopentaose V (LNnFP V), lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto- N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N-hexaose, difucosyl- lacto-N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH II), difucosyl-para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N-trifucoheptaose.

[0064] A 'fucosylated oligosaccharide' as used herein and as generally understood in the state of the art is an oligosaccharide that is carrying a fucose-residue. Such fucosylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a fucose. A fucosylated oligosaccharide can contain more than one fucose residue, e.g., two, three or more. A fucosylated oligosaccharide can be a neutral oligosaccharide or a charged oligosaccharide e.g., also comprising sialic acid structures. Fucose can be linked to other monosaccharide subunits comprising glucose, galactose, GIcNAc via alpha-glycosidic bonds comprising alpha-1,2 alpha-1,3, alpha-1,4, alpha-1,6 linkages. Examples comprise 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), Lacto-N- fucopentaose I (LNFP I), Lacto-N-fucopentaose II (LNFP II), Lacto-N-fucopentaose III (LNFP III), lacto-N- fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto-N-neofucopentaose I, lacto-N- difucohexaose I (LDFH I), lacto-N-difucohexaose II (LDFH II), Monofucosyllacto-N-hexaose III (MFLNH III), Difucosyllacto-N-hexaose (DFLNHa), difucosyl-lacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyl lacto-N- fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose.

[0065] Mammalian milk oligosaccharides or MMOs comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans (i.e. human milk oligosaccharides or HMOs) and mammals including but not limited to cows (Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Equusferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Loxodonta africana), giant anteater (Myrmecophaga tridactyla), common bottlenose dolphins (Tursiops truncates), northern minke whales (Balaenoptera acutorostrata), tammar wallabies (Macropus eugenii), red kangaroos (Macropus rufus), common brushtail possum (Trichosurus Vulpecula), koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus), platypus (Ornithorhynchus anatinus). As used herein, "mammalian milk oligosaccharide" or MMO refers to oligosaccharides such as but not limited to 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2', 3- difucosyllactose, 2',2-difucosyllactose, 3,4-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6- disialyllactose, 6,6'-disialyllactose, 8,3-disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyl lacto-N- tetraose b, sialyllacto-N-tetraose a, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosylmonosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto-N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N- hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated oligosaccharides, non-charged (neutral) oligosaccharide and / or sialylated oligosaccharides. As used herein, "mammalian milk oligosaccharide" refers to oligosaccharides such as but not limited to 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2',3-difucosyllactose, 2',2- difucosyllactose, 3,4-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6-disialyllactose, 6,6'- disialyllactose, 8,3-disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N- tetraose a, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para- lacto-N-hexaose, monofucosylmonosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N- hexaose I, sialyllacto-N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated oligosaccharides, non-charged (neutral) oligosaccharide and / or sialylated oligosaccharides.

[0066] The terms "LNT II", "LNT-II", "LN3", "lacto-N-triose II", "lacto- / V-triose II", "lacto-N-triose", "lacto- / V-triose" or "GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably. The terms "LNT", "lacto-N-tetraose", "lacto- / V-tetraose" or "Gaipi-3GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably. The terms "LNnT", "lacto-N-neotetraose", "lacto- / V-neotetraose", "neo-LNT" or "Gaipi-4GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably. The term "transporter protein" as used herein refers to a protein that is part of or interacts with the cell membrane and controls the flow of molecules and information across the cell. The transporter protein is thus involved in transport, be it import into or export out of the cell. Such transporter protein can be but is not limited to porters, P-P-bond-hydrolysis-driven transporters, P-Barrel Porins, auxiliary transport proteins and phosphotransfer-driven group translocators (Forrest et al., Biochim. Biophys. Acta 1807 (2011) 167-188; Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93; Moraes and Reithmeier, Biochim. Biophys. Acta 1818 (2012), 2687-2706; Saier et al., Nucleic Acids Res. 44 (2016) D372-D379).

[0067] The term "pathway for production of a saccharide" as used herein is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a saccharide as defined herein. Said pathway for production of a saccharide can comprise but is not limited to pathways involved in the synthesis of a nucleotide-activated sugar and the transfer of said nucleotide-activated sugar to a precursor to create a saccharide of the present invention. Examples of such pathways comprise but are not limited to a fucosylation, sialylation, galactosylation, N-acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation, N-acetylmannosaminylation pathway.

[0068] "Expression" of a membrane protein is defined as "overexpression" of the gene encoding said membrane protein in the case said gene is an endogenous gene or "expression" in the case the gene encoding said transporter protein is a homologous or heterologous gene that is not present in the wild-type strain or cell and is first introduced in said cell.

[0069] The term "purified" refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule. For cells, saccharides, nucleic acids, and polypeptides, the term "purified" refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state. Typically, purified saccharides, oligosaccharides, proteins or nucleic acids of the invention are at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 % or 85 % pure, usually at least about 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99.0 % pure as measured by band intensity on a silver-stained gel or other method for determining purity. Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized. For di- and oligosaccharides, purity can be determined using methods such as but not limited to thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis or mass spectroscopy. Further herein, the terms "contaminants" and "impurities" preferably mean particulates, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids and / or endotoxins which can be present in an aqueous medium like e.g., a cultivation or an incubation.

[0070] The term "clarifying" as used herein refers to the act of treating an aqueous medium like e.g., a cultivation or an incubation, to remove suspended particulates and contaminants from the production process, like e.g., cells, cell components, insoluble metabolites and debris, that could interfere with the eventual purification of the saccharide. Such treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional ultrasonic treatment, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g., through a ceramic filter which can include a Celite™ filter aid).

[0071] The term "cultivation" refers to the culture medium wherein the cell is cultivated, or fermented, the cell itself, and a saccharide that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell. The terms "culture medium" and "cultivation medium" as used herein are used interchangeably and refer to the medium wherein the cell is cultivated.

[0072] The term "incubation" refers to a mixture wherein a saccharide is produced. Said mixture can comprise one or more enzyme(s) and one or more precursor(s) as defined herein present in a buffered solution and incubated for a certain time at a certain temperature enabling production of a saccharide, catalysed by said one or more enzyme(s) using said one or more precursor(s) in said mixture. Said mixture can also comprise i) the cell obtained after cultivation or incubation, optionally said cell is subjected to cell lysis, ii) a buffered solution or the cultivation or incubation medium wherein the cell was cultivated or fermented, and iii) a saccharide that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell. Said incubation can also be the cultivation as defined herein.

[0073] The terms "reactor" and "incubator" refer to the recipient filled with the cultivation or incubation. Examples of reactors and incubators comprise but are not limited to microfluidic devices, well plates, tubes, shake flasks, fermenters, bioreactors, process vessels, cell culture incubators, CO2 incubators.

[0074] As used herein, the term "cell productivity index (CPI)" refers to the mass of the saccharide produced by the cells divided by the mass of the cells produced in the culture.

[0075] The term "precursor" as used herein refers to substances which are taken up or synthetized by the cell for the specific production of a saccharide according to the present invention. In this sense a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, which is first modified within the cell as part of the biochemical synthesis route of a saccharide. The term "precursor" as used herein is also to be understood as a chemical compound that participates in a chemical or enzymatic reaction to produce another compound like e.g. an intermediate or an acceptor as defined herein, as part in the metabolic pathway of a saccharide. The term "precursor" as used herein is also to be understood as a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond, as part in the metabolic pathway of a saccharide. Examples of such precursors used in the production of a saccharide comprise the acceptors as defined herein, and / or dihydroxyacetone, glucosamine, sialic acid, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates like e.g. but not limited to glucose-l-phosphate, galactose-l-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-l,6-bisphosphate, mannose-6-phosphate, mannose-l-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N- acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-1- phosphate, N-acetylneuraminic acid-9-phosphate and nucleotide-activated sugars like nucleotide diphospho-sugars and nucleotide monophospho-sugars as defined herein like e.g. UDP-glucose, UDP- galactose, UDP-N-acetylglucosamine, GDP-mannose, GDP-4-dehydro-6-deoxy-a-D-mannose, GDP- fucose, CMP-sialic acid, CMP-KDO.

[0076] Optionally, the cell is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the group consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the saccharide of present invention.

[0077] The term "acceptor" as used herein refers to a mono-, di- or oligosaccharide, which can be modified by a glycosyltransferase and that is used in the production of a saccharide. Examples of such acceptors comprise glucose, galactose, fructose, glycerol, fucose, mannose, maltose, sucrose, lactose, sialic acid, sialylated oligosaccharide, fucosylated oligosaccharide, lacto-N-triose, lacto-N-tetraose (LNT), lacto-N- neotetraose (LNnT), lacto-N-pentaose (LNP), lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N- neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para lacto-N- neohexaose (pLNnH), para lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N-neoheptaose, para lacto- N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, and oligosaccharide containing 1 or more N-acetyllactosamine units and / or 1 or more lacto-N-biose units or an intermediate into oligosaccharide and fucosylated versions thereof, ceramide, N-acylated sphingoid, glucosylceramide, lactosylceramide, sphingosine, phytosphingosine, sphingosine synthons, peptide backbones with beta-GIcNAc-Asn residues, glycoproteins with terminal GIcNAc and Gal residues, immunoglobulins.

[0078] Detailed description of the invention

[0079] According to a first aspect, the present invention provides a method for importing a disaccharide inside a cell, wherein the disaccharide is selected from the list consisting of or consisting essentially of lactose, lactulose, N-acetyllactosamine and lacto-N-biose. The method comprises (1) providing a cell, (2) metabolically engineering the cell by (i) introducing and expressing and / or( ii) overexpressing a membrane protein and (3) cultivating and / or incubating said cell, preferably in a cultivation medium comprising the disaccharide, preferably a single cell, under conditions permissive to express the membrane protein and to import the disaccharide inside the cell. The membrane protein has disaccharide importing activity across the membrane of said cell, originates from the major facilitator superfamily (MFS) of transporters and comprises a polypeptide sequence comprising (1) the conserved domain CD-INB1 with sequence [AGSV][HKNQR][CDEGNQTV] wherein the second amino acid residue is aligned to Lys 18 in SEQ ID NO 01, and (2) the conserved domain CD-INB2 with sequence [DEQ]XXX[FWY] wherein the first amino acid residue is aligned to Aspl24 in SEQ ID NO 01 and wherein X can be any amino acid residue.

[0080] According to a second aspect, the present invention provides a cell genetically engineered for the production of a saccharide, preferably a non-charged (neutral) oligosaccharide, wherein the cell comprises a pathway for production of said saccharide and is also genetically engineered to overexpress a membrane protein that (1) originates from the major facilitator superfamily (MFS) of transporters and that (2) is capable to transfer and / or transfers a disaccharide having a galactose (Gal) residue at its nonreducing end across the membrane of said cell. Herein, the membrane protein comprises a polypeptide sequence comprising (1) the conserved domain CD-INB1 with sequence [AGSV][HKNQR][CDEGNQTV] wherein the second amino acid residue is aligned to Lys 18 in SEQ ID NO 01, and (2) the conserved domain CD-INB2 with sequence [DEQ]XXX[FWY] wherein the first amino acid residue is aligned to Aspl24 in SEQ ID NO 01 and wherein X can be any amino acid residue.

[0081] According to a third aspect, the present invention provides a method for the production of a saccharide, preferably an oligosaccharide or a non-charged (neutral) oligosaccharide, more preferably a milk oligosaccharide, even more preferably a mammalian milk oligosaccharide, even more preferably a human milk oligosaccharide wherein the method comprises cultivating and / or incubating a cell, preferably a single cell, as described herein, in cultivation and / or incubation medium under conditions permissive to express a membrane protein as described herein and to produce said saccharide. According to this third aspect, in one embodiment, the method for importing a disaccharide inside a cell comprises a cell which is metabolically engineered to express the membrane protein as described herein and further comprises and / or is genetically engineered to comprise a pathway for production of a saccharide.

[0082] In the scope of the present invention, permissive conditions are understood to be conditions relating to physical or chemical parameters including but not limited to temperature, pH, pressure, osmotic pressure and product / precursor concentration.

[0083] In a particular embodiment, the permissive conditions may include a temperature-range of 30 + / - 20 degrees centigrade, a pH-range of 7 + / - 3.

[0084] In a specific embodiment, the cell comprises a pathway for production of a saccharide. The term "pathway for production of a saccharide" as used herein is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a saccharide as defined herein. Said pathway for production of a saccharide can comprise but is not limited to pathways involved in the synthesis of a nucleotide-activated sugar and the transfer of said nucleotide-activated sugar to a precursor to create a saccharide of the present invention. Examples of such pathways comprise but are not limited to a fucosylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N- acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway.

[0085] In another additional specific embodiment, the cell is genetically engineered to overexpress a membrane protein as described herein.

[0086] In a specific embodiment, the cell lacks a gene encoding for a lactose permease. In an alternative specific embodiment, the cell possesses and / or expresses at least one gene encoding for a lactose permease wherein said at least one gene encoding for a lactose permease is rendered less functional or is knocked out. In the scope of present invention, it is to be understood that present invention also covers a cell which possesses and / or expresses at least one gene encoding for a lactose permease wherein said at least one gene encoding for a lactose permease is first knocked out from said cell and then is re-introduced in said cell via means well-known in the art for expression and / or overexpression.

[0087] In the scope of present invention, said lactose permease is a protein that has transfer activity on betagalactosides across the membrane of the cell with the concomitant import of a proton (H+). The transfer activity of the lactose permease as described herein on beta-galactosides is any one of i) influx, ii) efflux or iii) influx and efflux of said beta-galactosides across the membrane of the cell. Preferably, the transfer activity of the lactose permease as described herein is influx of beta-galactosides into the cell. More preferably, the lactose permease as described herein is a proton symporter and can import, amongst others, lactose, melibiose, lactulose, methyl-l-thio-beta-D-galactopyranoside (TMG), isopropyl-p- thiogalactoside (IPTG), 4-nitrophenyl-beta-D-galactopyranoside, 4-nitrophenyl-alpha-D- galactopyranoside and galactopyranosyl-l-glycerol with the concomitant import of a proton (H+). Specifically, the lactose permease is capable of lactose influx and / or imports lactose in said cell. In the scope of present invention, the terms "lactose permease", "lactose transporter", "lacY", "LacY", "E II Lac", "lacEF", "P-galactopyranoside:H+ symporter", "galactoside permease" and "lactose / galactose transporter" are used interchangeably.

[0088] According to the method and / or cell of present invention, the lactose permease comprises a polypeptide sequence comprising the IPR000576 domain as defined by InterPro 98.0 as released on 25thJanuary 2024. Preferably, the lactose permease is part of the major facilitator superfamily (MFS) of transporters and is capable to transfer and / or transfers beta-galactosides across the membrane of said cell with proton symport. The major facilitator superfamily (MFS) is a superfamily of membrane transport proteins catalyzing uniport, solutexation (H+, but seldom Na+) symport and / or solute:H+ or solute:solute antiport. Most are of 400-600 amino acyl residues in length and possess either 12, 14, or occasionally, 24 transmembrane a-helical spanners (TMSs) (Reddy et al., Febs J. 2012, 279(11): 2022-2035).

[0089] In a more preferred embodiment of the method and / or cell of the invention, the lactose permease as described herein is LacY. In an even more preferred embodiment of the method and / or cell of the invention, the lactose permease is LacY from E. coli with UniProt ID P02920 or LacY from Citrobacter werkmanii with UniProt ID A0A6I5AGA6.

[0090] In another additional specific embodiment, the cell possesses and / or expresses at least one gene encoding for a lactose permease wherein said at least one gene encoding for a lactose permease is rendered less functional or is knocked out compared to a non-modified progenitor cell. Rendering a gene less functional is to be understood as rendering a gene less-able, i.e., statistically significantly 'less-able' compared to a functional wild-type gene or completely unable (such as knocked-out genes) to produce a functional final product. A gene can be made less functional by means of common well-known technologies for a skilled person, by e.g., any one or more of insertion, deletion and / or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) chosen from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said gene so that said gene is made less-able to produce a functional final product. Methods like e.g. siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes and transposon mutagenesis could be used herein. A knock-out of a gene from the cell's genome can be made by methods well-known by a person skilled in the art.

[0091] In a preferred embodiment of the method and / or cell of present invention, the at least one gene encoding for a lactose permease is rendered less functional by insertion, deletion and / or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list consisting of or consisting essentially of promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said at least one gene.

[0092] In a preferred embodiment of the method and / or cell of the invention, the metabolically engineered cell is modified with gene overexpression modules for a membrane protein as described herein. In a preferred embodiment of the method and / or cell of present invention, the cell is genetically engineered to express and / or overexpress a membrane protein as described herein. Preferably, the genetically engineered cell is modified with gene expression modules wherein the expression from any one of said expression modules is constitutive or is tuneable.

[0093] In a preferred embodiment of the method and / or cell of the invention, the genetically engineered cell is modified with gene expression modules for proteins other than a membrane protein as described herein wherein the expression from any of said expression modules is constitutive or is tuneable.

[0094] Said expression and overexpression modules are also known as transcriptional units and comprise polynucleotides for expression and overexpression, respectively, of recombinant genes including coding gene sequences and appropriate transcriptional and / or translational control signals that are operably linked to the coding genes. Said control signals comprise promoter sequences, untranslated regions, ribosome binding sites, terminator sequences. Said expression and overexpression modules can contain elements for expression and overexpression, respectively, of one single recombinant gene but can also contain elements for expression and overexpression, respectively, of more recombinant genes or can be organized in an operon structure for integrated expression and overexpression, respectively, of two or more recombinant genes. Said polynucleotides may be produced by recombinant DNA technology using techniques well-known in the art. Methods which are well known to those skilled in the art to construct expression and overexpression modules include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and yearly updates).

[0095] According to a preferred embodiment of the present invention, the cell is modified with one or more expression modules or overexpression modules for a membrane protein as described herein. According to a preferred embodiment of the present invention, the cell is also modified with one or more expression modules for one or more protein(s) different from a membrane protein as described herein. The expression and / or overexpression modules can be integrated in the genome of said cell or can be presented to said cell on a vector. Said vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed / transfected into said metabolically engineered cell. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense / antisense markers. The expression and / or overexpression system constructs may contain control regions that regulate as well as engender expression and / or overexpression. Generally, any system or vector suitable to maintain, propagate, express or overexpress polynucleotides and / or to express and / or overexpress a polypeptide in a host may be used for expression and / or overexpression in this regard. The appropriate DNA sequence may be inserted into the expression and / or overexpression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., see above. For recombinant production, cells can be genetically engineered to incorporate expression and / or overexpression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986), and Sambrook et al., 1989, supra.

[0096] As used herein an expression module or an overexpression module comprises polynucleotides for expression or overexpression of at least one recombinant gene. Said recombinant gene is involved in the pathway for production of a saccharide; or said recombinant gene is linked to other pathways in said cell that are not involved in the synthesis of a saccharide. Said recombinant genes encode endogenous proteins with a modified expression or activity, preferably said endogenous proteins are overexpressed; or said recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed in said modified cell, preferably overexpressed. The endogenous proteins can have a modified expression in the cell which also expresses a heterologous protein. Said recombinant genes may encode endogenous proteins that have been adapted for overexpression; or said recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed or overexpressed in said modified cell. The cell can be modified for overexpression for both one or more endogenous protein(s) and one or more heterologous protein(s).

[0097] According to a preferred embodiment of the present invention, the overexpression of each of said overexpression modules or the expression of each of said expression modules is constitutive or tuneable as defined herein.

[0098] In a preferred embodiment of the method and / or cell of the invention, the cell is modified in the expression or activity, and / or is modified for overexpression, of a membrane protein as described herein. Preferably, said membrane protein is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous membrane protein is overexpressed; alternatively said membrane protein is a heterologous protein that is heterogeneously introduced and expressed and / or overexpressed in said cell, preferably overexpressed. In another preferred embodiment of the method and / or cell of the invention, the cell is modified for expression and / or overexpression of one or more endogenous membrane protein(s) and / or one or more heterologous membrane protein(s).

[0099] According to present invention, the membrane protein originates from the major facilitator superfamily (MFS) of transporters and is capable to transfer and / or transfers a disaccharide, preferably having a galactose (Gal) residue at its non-reducing end, across the membrane of said cell, preferably inside said cell. The major facilitator superfamily (MFS) is a superfamily of membrane transport proteins catalyzing uniport, solutexation (H+, but seldom Na+) symport and / or solute:H+ or solute:solute antiport. Most are of 400-600 amino acyl residues in length and possess either 12, 14, or occasionally, 24 transmembrane a- helical spanners (TMSs) (Reddy et al., Febs J. 2012, 279(11): 2022-2035).

[0100] In a preferred embodiment of the method and / or cell of present invention, the transfer activity of the membrane protein as described herein on a disaccharide having a Gal residue at its non-reducing end is any one of i) influx, ii) efflux or iii) influx and efflux of said disaccharide across the membrane of said cell. In a more preferred embodiment, said transfer activity of said membrane protein is influx of said disaccharide having a Gal residue at its non-reducing end from the outside to in said cell.

[0101] In another and / or additional preferred embodiment of the method and / or cell of present invention, the membrane protein as described herein is capable to use and / or uses one or more coupling cation(s) for the transfer activity of said membrane protein on said disaccharide having a Gal residue at its nonreducing end, wherein said coupling cation is selected from the list comprising, consisting of or consisting essentially of H+, Li+and Na+.

[0102] In a preferred embodiment of the method and / or cell of present invention, said disaccharide having a Gal residue at its non-reducing end is selected from the list consisting of or consisting essentially of lactose, lactulose, melibiose, N-acetyllactosamine (LacNAc) and lacto-N-biose (LNB) as described herein. In a more preferred embodiment, said disaccharide having a Gal residue at its non-reducing end is melibiose.

[0103] In a preferred embodiment of the method and / or cell of present invention, said membrane protein is not capable to transfer and / or does not transfer said saccharide of present invention across the membrane of said cell.

[0104] In a preferred embodiment of the method and / or cell of present invention, the membrane protein comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001927, IPR018043, IPR039672 and IPR036259 domain as defined by InterPro 98.0 as released on 25thJanuary 2024 and is:

[0105] - any one of SEQ ID NOs 01, 02, 03, 04 or 05,

[0106] - a functional homolog or functional fragment of any one of said SEQ ID NOs 01, 02, 03, 04 or 05, or

[0107] - a sequence having at least 40 % sequence identity to any one of the full-length amino acid sequences as represented by any one of SEQ ID NOs 01, 02, 03, 04 or 05.

[0108] In another and / or additional preferred embodiment of the method and / or cell of present invention, the membrane protein comprises a polypeptide sequence comprising the conserved domain cdl7332 domain as defined by the Conserved Domain Database CDD 3.20 as released on 6thOctober 2022 and is:

[0109] - any one of SEQ ID NOs 01, 02, 03, 04 or 05,

[0110] - a functional homolog or functional fragment of any one of said SEQ ID NOs 01, 02, 03, 04 or 05, or

[0111] - a sequence having at least 40 % sequence identity to any one of the full-length amino acid sequences as represented by any one of SEQ ID NOs 01, 02, 03, 04 or 05. In another and / or additional preferred embodiment of the method and / or cell of present invention, the membrane protein comprises a polypeptide sequence comprising the PANTHER domain PTHR11328 as defined by PANTHER 18.0 as released on 17thSeptember 2023 and is:

[0112] - any one of SEQ ID NOs 01, 02, 03, 04 or 05,

[0113] - a functional homolog or functional fragment of any one of said SEQ ID NOs 01, 02, 03, 04 or 05, or

[0114] - a sequence having at least 40 % sequence identity to any one of the full-length amino acid sequences as represented by any one of SEQ ID NOs 01, 02, 03, 04 or 05.

[0115] In another and / or additional preferred embodiment of the method and / or cell of present invention, the membrane protein comprises a polypeptide sequence comprising the PFAM domain PF13347 as defined by PFAM 32.0 as released in Sept 2018 and is:

[0116] - any one of SEQ ID NOs 01, 02, 03, 04 or 05,

[0117] - a functional homolog or functional fragment of any one of said SEQ ID NOs 01, 02, 03, 04 or 05, or

[0118] - a sequence having at least 40 % sequence identity to any one of the full-length amino acid sequences as represented by any one of SEQ ID NOs 01, 02, 03, 04 or 05.

[0119] In a preferred embodiment of the method and / or cell of present invention, said membrane protein is any one of an endogenous protein, a homologous protein or a heterologous protein of said cell.

[0120] In a preferred embodiment of the method and / or cell of present invention, the cell comprises a polynucleotide sequence which encodes a membrane protein as described herein. In a more preferred embodiment, the nucleic acid molecule is operably linked to control sequences recognized by the cell, said nucleic acid molecule further i) being integrated in the genome of said cell and / or ii) presented to said cell on a vector. In an additional and / or alternative more preferred embodiment, the nucleic acid molecule is foreign to said cell.

[0121] In a specific embodiment of the method of the present invention, the cell further comprises a pathway for production of a saccharide, preferably an oligosaccharide, more preferably a milk oligosaccharide, even more preferably a mammalian milk oligosaccharide or even more preferably a human milk oligosaccharide. This method comprises a step of cultivating and / or incubating said cell under conditions permissive to express said membrane protein and to produce said saccharide.

[0122] In a specific embodiment of the method and / or cell of present invention, the cell is genetically engineered for production of a saccharide, wherein the cell comprises a pathway for production of said saccharide. In a preferred embodiment, the cell is genetically engineered for production of two or more saccharides. In another and / or additional preferred embodiment, the cell is genetically engineered for an enhanced production of a saccharide, an enhanced uptake of one or more precursor(s) that is / are used in the synthesis of a saccharide, a better efflux of the saccharide, a decreased production of by-products that are synthesised together with the saccharide, an increased availability of co-factors like e.g. ATP, NADP, NADPH, and / or better metabolic flux through any one of the sialylation, fucosylation, galactosylation, N- acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation, and / or N- acetylmannosaminylation pathway present in the cell.

[0123] In a preferred embodiment of the method and / or cell of the present invention, the pathway for production of a saccharide as described herein is selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N- acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway.

[0124] In a preferred embodiment, the cell is genetically engineered to comprise a fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N- acetylgalactosaminylation pathway, mannosylation pathway and / or N-acetylmannosaminylation pathway for production of a saccharide as described herein. In an even more preferred embodiment, the cell comprises a fucosylation pathway, sialylation pathway, galactosylation pathway, N- acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and / or N-acetylmannosaminylation pathway wherein said fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and / or N-acetylmannosaminylation pathway has / have been genetically engineered for production of a saccharide as described herein.

[0125] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell comprises at least one pathway selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway wherein at least one of said pathway(s) has / have been genetically engineered for production of a saccharide as described herein.

[0126] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell further comprises a pathway selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway. In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell is genetically engineered to further comprise a pathway selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N- acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway. In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell further comprises at least one pathway selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway wherein at least one of said pathway(s) has / have been genetically engineered. Preferably, the cell further comprises a pathway selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N- acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway wherein any one of said fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway and preferably is used in said cell for production of a mixture of oligosaccharides, preferably of a non-charged (neutral) oligosaccharide and one or more other oligosaccharide(s) wherein said other oligosaccharide(s) is / are selected from the list comprising charged and non-charged (neutral) oligosaccharide.

[0127] In another and / or additional preferred embodiment of the method and / or cell, the cell further comprises a fucosylation pathway. In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to comprise a fucosylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been genetically engineered to comprise a fucosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-1- phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase and fucosyltransferase has / have a modified and / or enhanced expression.

[0128] In another and / or additional preferred embodiment of the method and / or cell, the cell further comprises a sialylation pathway. In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to comprise a sialylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been genetically engineered to comprise a sialylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N- acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GIcNAc 2- epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, d-arabinose 5- phosphate isomerase, KDO-8P synthase, KDO 8-phosphate phosphatase, CMP-KDO synthetase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase and sialyltransferase has / have a modified and / or enhanced expression.

[0129] In another and / or additional preferred embodiment of the method and / or cell, the cell further comprises a galactosylation pathway. In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to comprise a galactosylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been genetically engineered to comprise a galactosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase and galactosyltransferase has / have a modified and / or enhanced expression.

[0130] In another and / or additional preferred embodiment of the method and / or cell, the cell further comprises an 'N-acetylglucosaminylation' pathway. In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to comprise an N-acetylglucosaminylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been genetically engineered to comprise an N-acetylglucosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N- acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase, and a glycosyltransferase transferring GIcNAc has / have a modified and / or enhanced expression.

[0131] In another and / or additional preferred embodiment of the method and / or cell, the cell further comprises an 'N-acetylgalactosaminylation' pathway. In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to comprise an N-acetylgalactosaminylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been genetically engineered to comprise an N-acetylgalactosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6- phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-1- phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase, UDP-N-acetylglucosamine 4- epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase and / or UDP-N-acetylgalactosamine pyrophosphorylase and a glycosyltransferase transferring GalNAc has / have a modified and / or enhanced expression.

[0132] In another and / or additional preferred embodiment of the method and / or cell, the cell further comprises a 'mannosylation' pathway. In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to comprise a mannosylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been genetically engineered to comprise a mannosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase and / or mannose-1- phosphate guanylyltransferase and mannosyltransferase has / have a modified and / or enhanced expression.

[0133] In another and / or additional preferred embodiment of the method and / or cell, the cell further comprises an 'N-acetylmannosaminylation' pathway. In a more preferred additional and / or alternative embodiment, the cell is genetically engineered to comprise an N-acetylmannosaminylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been genetically engineered to comprise an N-acetylmannosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N- acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase, UDP-GIcNAc 2-epimerase and / or ManNAc kinase and a glycosyltransferase transferring ManNAc has / have a modified and / or enhanced expression.

[0134] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell further comprises one or more pathway(s) for monosaccharide synthesis. Said pathways for monosaccharide synthesis comprise enzymes like e.g. carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases, enzymes involved in the synthesis of one or more nucleoside triphosphate(s) like UTP, GTP, ATP and CTP, enzymes involved in the synthesis of any one or more nucleoside mono- or diphosphates like e.g. UMP and UDP, respectively, and enzymes involved in the synthesis of phosphoenolpyruvate (PEP).

[0135] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell further comprises one or more pathway(s) for phosphorylated monosaccharide synthesis. Said pathways for phosphorylated monosaccharide synthesis comprise enzymes involved in the synthesis of one or more monosaccharide(s), one or more nucleoside mono-, di- and / or triphosphate(s) and enzymes involved in the synthesis of phosphoenolpyruvate (PEP) like e.g. but not limited to PEP synthase, carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases and dehydrogenases. In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell further comprises one or more pathways for the synthesis of one or more nucleotide-activated sugars. Said pathways for nucleotide-activated sugar synthesis comprise enzymes like e.g. PEP synthase, carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases, mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, L-fucokinase / GDP-fucose pyrophosphorylase, L-glutamine— D-fructose-6- phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N- acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylglucosamine-6P 2-epimerase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine-6-phosphate phosphatase, N- acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-1- phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase, sialic acid synthase, N- acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphate phosphatase, d-arabinose 5-phosphate isomerase, KDO-8P synthase, KDO 8-phosphate phosphatase, CMP-KDO synthetase, CMP-sialic acid synthase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, glucophosphomutase and / or N-acetylglucosamine-l-phosphate uridylyltransferase.

[0136] In another and / or additional preferred embodiment of the method and / or cell, the cell further possesses, preferably expresses, more preferably overexpresses, one or more glycosyltransferase(s) selected from the list comprising, consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N- acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino- 4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP- / V-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.

[0137] In a more preferred embodiment of the method and / or cell of the invention, the fucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase and alpha-1, 6-fucosyltransferase. In an alternative and / or additional more preferred embodiment of the method and / or cell of the invention, the further sialyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase, and alpha-2, 8-sialyltransferase.

[0138] In an alternative and / or additional more preferred embodiment of the method and / or cell of the invention, the galactosyltransferase is selected from the list comprising, consisting of or consisting essentially of beta-1, 3-galactosyltransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, beta- 1,4-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3- galactosyltransferase and alpha-1, 4-galactosyltransferase.

[0139] In an alternative and / or additional more preferred embodiment of the method and / or cell of the invention, the glucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-glucosyltransferase, beta-1, 2-glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase.

[0140] In an alternative and / or additional more preferred embodiment of the method and / or cell of the invention, the mannosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, 2-mannosyltransferase, alpha-1, 3-mannosyltransferase and alpha-1, 6- mannosyltransferase.

[0141] In an alternative and / or additional more preferred embodiment of the method and / or cell of the invention, the N-acetylglucosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of galactoside beta-1, 3-N-acetylglucosaminyltransferase and beta-1, 6-N- acetylglucosaminyltransferase.

[0142] In an alternative and / or additional more preferred embodiment of the method and / or cell of the invention, the N-acetylgalactosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, 3-N-acetylgalactosaminyltransferase.

[0143] In an alternative and / or additional more preferred embodiment of the method and / or cell of the invention, the cell is modified in the expression or activity of at least one of said glycosyltransferases. In a preferred embodiment, said glycosyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous glycosyltransferase is overexpressed; alternatively said glycosyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed. Said endogenous glycosyltransferase can have a modified expression in the cell which also expresses a heterologous glycosyltransferase.

[0144] In another and / or additional preferred embodiment of the method and / or cell, the cell is further capable to produce, preferably produces, one or more nucleotide-activated sugars, preferably said cell is genetically engineered for production of one or more of said nucleotide-activated sugar(s). Herein, said one or more nucleotide-activated sugar(s) is / are selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP- GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2- acetamido-2,6-dideoxy--L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N- acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N- acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L- galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6- dideoxy-L-glucose), CMP-sialic acid, CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP- Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP- 2-keto-3-deoxymanno-octulonic acid (CMP-KDO), GDP-rhamnose and UDP-xylose.

[0145] In another and / or additional preferred embodiment of the method and / or cell, the cell further comprises a pathway for the synthesis of a nucleotide-activated sugar selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP- GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2- acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N- acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N- acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L- galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6- dideoxy-L-glucose), CMP-sialic acid, CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP- Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP- 2-keto-3-deoxymanno-octulonic acid (CMP-KDO), GDP-rhamnose and UDP-xylose.

[0146] The cell used herein is optionally genetically modified to express genes for the de novo synthesis of UDP- GIcNAc. UDP-GIcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing an UDP-GIcNAc can express enzymes converting, e.g. GIcNAc, which is to be added to the cell, to UDP-GIcNAc. These enzymes may be any one or more of the list comprising an N-acetyl-D- glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, and a bifunctional N-acetylglucosamine-l-phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli. Preferably, the cell is modified to produce UDP-GIcNAc. Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of CMP-Neu5Ac. CMP-Neu5Ac can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing CMP-Neu5Ac can express an enzyme converting, e.g., sialic acid to CMP-Neu5Ac. This enzyme may be a CMP-sialic acid synthetase, like the N-acylneuraminate cytidylyltransferase from several species including Homo sapiens, Neisseria meningitidis, and Pasteurella multocida. Preferably, the cell is modified to produce CMP- Neu5Ac. More preferably, the cell is modified for enhanced CMP-Neu5Ac production. Said modification can be any one or more selected from the list comprising knock-out of an N-acetylglucosamine-6- phosphate deacetylase, knock-out of a glucosamine-6-phosphate deaminase, over-expression of a CMP- sialic acid synthetase, and over-expression of an N-acetyl-D-glucosamine-2-epimerase encoding gene. Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of GDP-fucose. GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing GDP-fucose can express an enzyme converting, e.g., fucose, which is to be added to the cell, to GDP-fucose. This enzyme may be, e.g., a bifunctional fucose kinase / fucose-l-phosphate guanylyltransferase, like Fkp from Bacteroidesfragilis, or the combination of one separate fucose kinase together with one separate fucose-l-phosphate guanylyltransferase like they are known from several species including Homo sapiens, Sus scrofa and Rattus norvegicus. Preferably, the cell is modified to produce GDP-fucose. More preferably, the cell is modified for enhanced GDP-fucose production. Said modification can be any one or more selected from the list comprising knock-out of an UDP-glucose:undecaprenyl-phosphate glucose-l-phosphate transferase encoding gene, over-expression of a GDP-L-fucose synthase encoding gene, over-expression of a GDP-mannose 4,6-dehydratase encoding gene, over-expression of a mannose-l-phosphate guanylyltransferase encoding gene, over-expression of a phosphomannomutase encoding gene and over-expression of a mannose-6-phosphate isomerase encoding gene. Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-Gal. UDP-Gal can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing UDP-Gal can express an enzyme converting, e.g. UDP-glucose, to UDP-Gal. This enzyme may be, e.g., the UDP-glucose-4-epimerase GalE like as known from several species including Homo sapiens, Escherichia coli, and Rattus norvegicus. Preferably, the cell is modified to produce UDP-Gal. More preferably, the cell is modified for enhanced UDP-Gal production. Said modification can be any one or more selected from the list comprising knock-out of a bifunctional 5'- nucleotidase / UDP-sugar hydrolase encoding gene, knock-out of a galactose-l-phosphate uridylyltransferase encoding gene and over-expression of an UDP-glucose-4-epimerase encoding gene. Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-GalNAc. UDP-GalNAc can be synthesized from UDP-GIcNAc by the action of a single-step reaction using an UDP-N-acetylglucosamine 4-epimerase like e.g. wbgU from Plesiomonas shigelloides, gne from Yersinia enterocolitica or wbpP from Pseudomonas aeruginosa serotype 06. Preferably, the cell is modified to produce UDP-GalNAc. More preferably, the cell is modified for enhanced UDP-GalNAc production. Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-ManNAc. UDP-ManNAc can be synthesized directly from UDP-GIcNAc via an epimerization reaction performed by an UDP-GIcNAc 2-epimerase (like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica). Preferably, the cell is modified to produce UDP-ManNAc. More preferably, the cell is modified for enhanced UDP-ManNAc production.

[0147] In another and / or additional preferred embodiment of the method and / or cell, the cell possesses, preferably expresses, more preferably overexpresses one or more genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L-glutamine— D-fructose-6- phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-epimerase, UDP-GIcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N- acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase, Neu5Ac synthase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate- 9-phosphate synthase, N-acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, d-arabinose 5-phosphate isomerase, KDO-8P synthase, KDO 8-phosphate phosphatase, CMP-KDO synthetase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N- acetylgalactosamine pyrophosphorylase.

[0148] In another and / or additional preferred embodiment of the method and / or cell, the cell further comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated. These mono-, di-, or oligosaccharides are involved in and / or required for the synthesis of said saccharide.

[0149] In a preferred embodiment of the method and / or cell of present invention, the saccharide is selected from the list consisting of or consisting essentially of monosaccharide; phosphorylated monosaccharide; activated monosaccharide; disaccharide; oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); sialylated milk oligosaccharide; neutral (noncharged) milk oligosaccharide; fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; O-antigen; enterobacterial common antigen (ECA); the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; an antigen of the human ABO blood group system; an animal oligosaccharide; animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide; plant oligosaccharide selected from the list consisting of N-glycans and O-glycans; fucosylated oligosaccharide; fucosylated oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose ll7lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose; sialylated oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N- tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'- sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto- N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N- acetylglucosamine containing neutral (non-charged) saccharide; N-acetylglucosamine containing neutral (non-charged) saccharide selected from the list comprising lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6' -galactosyllactose, 3' -galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N- neohexaose, fucosyl-lacto-N-hexaose, difucosyl-lacto-N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH II), difucosyl-para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N- trifucoheptaose; N-acetylglucosamine containing saccharide; N-acetyllactosamine containing saccharide; lacto-N-biose containing saccharide; non-fucosylated neutral (non-charged) saccharide; chitosan; chitosan comprising oligosaccharide; heparosan; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan sulphate; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.

[0150] In a more preferred embodiment, the saccharide is a mammalian milk oligosaccharide as described herein. In another more preferred embodiment, the saccharide is a human milk oligosaccharide as described herein. In another more preferred embodiment, the saccharide is an animal saccharide selected from the list consisting of N-glycans and O-glycans. In another more preferred embodiment, the saccharide is a plant saccharide selected from the list consisting of N-glycans and O-glycans. In the context of present invention, N-glycans and O-glycans refer to the oligosaccharide structures as known by the person skilled in the art wherein said structures are not attached to a protein or a peptide.

[0151] In even more preferred embodiment, the saccharide is selected from the list comprising, consisting of or consisting essentially of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6- fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose ll7lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N- hexaose, difucosyl-lacto-N-neohexaose, 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N- hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N-hexaose, difucosyl-lacto-N-hexaose, difucosyl-lacto- N-neohexaose (LNnDFH II), difucosyl-para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N- fucohexaose and lacto-N-trifucoheptaose.

[0152] In a preferred embodiment of the method and / or cell of present invention, the non-charged (neutral) oligosaccharide is an oligosaccharide selected from the list consisting of or consisting essentially of noncharged (neutral) milk oligosaccharide; non-charged (neutral) mammalian milk oligosaccharide (MMO); non-charged (neutral) human milk oligosaccharide (HMO); fucosylated non-charged (neutral) oligosaccharide; non-fucosylated non-charged (neutral) oligosaccharide; fucosylated non-charged (neutral) milk oligosaccharide; non-fucosylated non-charged (neutral) milk oligosaccharide; fucosylated non-charged (neutral) MMO; non-fucosylated non-charged (neutral) MMO; fucosylated non-charged (neutral) HMO; non-fucosylated non-charged (neutral) HMO; N-acetylglucosamine containing noncharged (neutral) oligosaccharide; N-acetylglucosamine containing non-charged (neutral) milk oligosaccharide; N-acetylglucosamine containing non-charged (neutral) MMO; N-acetylglucosamine containing non-charged (neutral) HMO; N-acetyllactosamine containing non-charged (neutral) oligosaccharide; lacto-N-biose containing non-charged (neutral) oligosaccharide; galactosylated noncharged (neutral) oligosaccharide; galactosylated non-charged (neutral) milk oligosaccharide; an antigen of the human ABO blood group system; O-antigen; enterobacterial common antigen (ECA); an animal non-charged (neutral) oligosaccharide; a plant non-charged (neutral) oligosaccharide; chitosan; chitosan comprising non-charged (neutral) oligosaccharide; fucosylated non-charged (neutral) oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6- fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose; N-acetylglucosamine containing non-charged (neutral) oligosaccharide selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N- neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N-hexaose, difucosyl-lacto-N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH 11), difucosyl-para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N-trifucoheptaose. In a more preferred embodiment, the noncharged (neutral) oligosaccharide is a mammalian milk oligosaccharide as described herein. In another more preferred embodiment, the non-charged (neutral) oligosaccharide is a human milk oligosaccharide as described herein. In another more preferred embodiment, the non-charged (neutral) oligosaccharide is an animal non-charged (neutral) oligosaccharide selected from the list consisting of N-glycans and O- glycans. In another more preferred embodiment, the non-charged (neutral) oligosaccharide is a plant noncharged (neutral) oligosaccharide selected from the list consisting of N-glycans and O-glycans. In the context of present invention, N-glycans and O-glycans refer to the oligosaccharide structures as known by the person skilled in the art wherein said structures are not attached to a protein or a peptide.

[0153] In even more preferred embodiment, the non-charged (neutral) oligosaccharide is selected from the list comprising, consisting of or consisting essentially of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4- fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N- neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N- fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose, difucosyl-lacto-N-neohexaose, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N- neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N-hexaose, difucosyl-lacto- N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH II), difucosyl-para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N-trifucoheptaose.

[0154] The saccharide or the oligosaccharide in the context of the present invention is preferably in free form, i.e., the saccharide or the oligosaccharide does not contain any protective group.

[0155] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell is capable to produce, preferably produces, said saccharide from one or more precursor(s) as defined herein. In a more preferred embodiment, the precursor is lactose. Preferably, said one or more precursor(s) is / are fed to the cell from the culture medium or the incubation. In another more preferred embodiment, the cell is capable to produce, preferably produces, at least one of said one or more precursor(s). In an even more preferred embodiment, the cell is capable to produce, preferably produces, all of said one or more precursor(s). In another more preferred embodiment, the cell is genetically engineered for the production of at least one of said one or more precursor(s). In an even more preferred embodiment, the cell is genetically engineered for the production of all of said one or more precursor(s). In another more preferred embodiment, at least one of said one or more precursor(s) is internalized in said cell via (1) one or more transporter protein(s) and / or (2) said membrane protein. In another more preferred embodiment, at least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s) or transporter protein(s). In another and / or additional more preferred embodiment, at least one of said one or more precursor(s) is internalized in said cell via a membrane protein as described herein. In another preferred embodiment, the precursor(s) that is / are used by the cell for the production of said saccharide is / are completely converted into said saccharide.

[0156] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell is selected from the list consisting of consisting of prokaryotic cells and eukaryotic cells, preferably from the list consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, plant cells, fungal cells, animal cells, insect cells and protozoan cells. In another preferred embodiment, the cell is a bacterium, fungus, yeast, a plant cell, an animal cell or a protozoan cell.

[0157] The latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus or the phylum of Actinobacteria. The latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains - designated as E. coli K12 strains - which are well- adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Hence, the present invention specifically relates to a mutated and / or transformed Escherichia coli cell or strain as indicated above wherein said E. coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is E. coli MG1655. The latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobacilliales, with members such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacil lales with members such as from the genus Bacillus, such as Bacillus subtilis or, B. amyloliquefaciens. The latter Bacterium belonging to the phylum Actinobacteria, preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. afermentans, or belonging to the family of the Streptomycetaceae with members Streptomyces griseus or S. fradiae. The latter bacterium belonging to the phylum Proteobacteria, preferably belonging to the family of the Vibrionaceae, with member Vibrio natriegens. The latter yeast preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes. The latter yeast belongs preferably to the genus Saccharomyces (with members like e.g. Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members like e.g. Pichia pastoris, P. anomala, P. kluyveri), Komagataella, Hansenula, Kluyveromyces (with members like e.g. Kluyveromyces lactis, K. marxianus, K. thermotolerans), Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces, Torulaspora, Yarrowia (like e.g. Yarrowia lipolytica) or Starmerella (like e.g. Starmerella bombicola). The latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolytica, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, Kluyveromyces marxianus, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii. The latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. Plant cells include cells of flowering and non-flowering plants, as well as algal cells, for example Chlamydomonas, Chlorella, etc. Preferably, said plant is a tobacco, rose, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant. More preferably, the latter plant cell is selected from the Rosa family. The latter animal cell is preferably derived from non-human mammals (e.g. cattle, buffalo, pig, sheep, mouse, rat, primate (e.g., chimpanzee, orangutan, gorilla, monkey (e.g., Old World, New World), lemur), dog, cat, rabbit, horse, cow, goat, ox, deer, musk deer, bovid, whale, dolphin, hippopotamus, elephant, rhinoceros, giraffe, zebra, lion, cheetah, tiger, panda, red panda, otter), birds (e.g. chicken, duck, ostrich, turkey, pheasant), fish (e.g. swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g. lobster, crab, shrimp, clams, oyster, mussel, sea urchin), reptiles (e.g. snake, alligator, turtle), amphibians (e.g. frogs) or insects (e.g. fly, nematode) or is a genetically modified cell line derived from human cells excluding embryonic stem cells. Both human and non-human mammalian cells are preferably selected from the list comprising an epithelial cell like e.g. a mammary epithelial cell, an embryonic kidney cell (e.g. HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g. an N20, SP2 / 0 or YB2 / 0 cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof such as described in WO21067641, preferably mesenchymal stem cell or derivates thereof as described in WO21067641, a lactocyte derived from mammalian induced pluripotent stem cells, preferably human induced pluripotent stem cells, a lactocyte as part of mammary-like gland organoids, a post-parturition mammary epithelium cell, a polarized mammary cell, preferably a polarized mammary cell selected from the list comprising live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells, live immortalized mammary epithelial cells, live immortalized mammary myoepithelial cells, live immortalized mammary progenitor cells, a non-mammary adult stem cell or derivatives thereof as well-known to the person skilled in the art from e.g. WO2021 / 219634, WO 2022 / 054053, WO 2021 / 141762, WO 2021 / 142241, WO 2021 / 067641 and WO2021 / 242866. The latter insect cell is preferably derived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cells or Drosophila melanogaster like e.g. Drosophila S2 cells. The latter protozoan cell preferably is a Leishmania tarentolae cell.

[0158] In another and / or additional preferred embodiment, the cell is an E. coll or yeast with a lactose permease positive phenotype, preferably wherein said lactose permease is coded by the gene LacY or LAC1Z, respectively.

[0159] In another and / or additional preferred embodiment, the cell is an E. coli or yeast with a lactose permease negative phenotype, preferably wherein said lactose permease is coded by the gene LacY or LAC12, respectively.

[0160] In another and / or additional preferred embodiment, the cell is an E. coli or yeast with a knocked-out or knocked-down lactose permease, preferably wherein said lactose permease is coded by the gene LacY or LAC12, respectively.

[0161] In a preferred embodiment of the method of present invention, the saccharide of present invention is produced by a cell that is cultured in a cell cultivation. Within the context of present invention, the cell cultivation comprises in vitro and / or ex vivo cultivation of cells. In another and / or additional more preferred embodiment, the cell cultivation is a fermentation. In an alternative and / or additional more preferred embodiment, the cell is cultivated or incubated in a reactor as defined herein. In an alternative and / or additional more preferred embodiment, the cell is cultivated or incubated in an incubator as defined herein.

[0162] In another and / or additional preferred embodiment of the method of present invention, the cell is cultivated in cultivation or incubation medium comprising at least one carbon source selected from the list comprising, consisting of or consisting essentially of a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract. Preferably, said carbon source is selected from the list comprising, consisting of or consisting essentially of glucose, N-acetylglucosamine (GIcNAc), glycerol, fructose, sucrose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate. More preferably, said at least one carbon source selected from the list consisting of glucose, fructose, sucrose, and glycerol. In a more preferred embodiment, the cultivation or incubation medium is a chemically defined medium. In an additional preferred embodiment, the cultivation or incubation medium is a minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and / or selenium. In another and / or additional preferred embodiment, the cultivation or incubation medium contains at least one compound selected from the list consisting of or consisting essentially of lactose, galactose, glucose, sialic acid, UDP-Gal, UDP-GIcNAc, UDP-GalNAc and CMP-sialic acid.

[0163] In another and / or additional preferred embodiment of the method of present invention, the cultivation or incubation medium comprises one or more precursor(s) that is / are used for production of said saccharide. In a more preferred embodiment, the precursor is selected from the list comprising, consisting of or consisting essentially of glucose, galactose, GIcNAc, GalNAc, sialic acid, UDP-GIcNAc, UDP-GalNAc, UDP-Gal, CMP-sialic acid, lactose, GlcNAc-pi,3-Gal-pi,4-Glc (LN3, lacto-N-triose), Gal-pi,3-GlcNAc-pi,3- Gal-pi,4-Glc (LNT, lacto-N-tetraose), and Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc (LNnT, lacto-N-neotetraose). In another and / or alternative preferred embodiment of the method of present invention, at least one precursor feed for the production of said saccharide is added to said cultivation or incubation medium.

[0164] Preferably, when performing the method as described herein, a first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the cultivation or incubation medium before the lactose is added to the cultivation or incubation medium in a second phase.

[0165] In an alternative preferable embodiment, in the method as described herein, the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.

[0166] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell produces 25 g / L or more of said saccharide in the whole broth and / or supernatant and / or wherein said saccharide in the whole broth and / or supernatant has a purity of at least 80 % measured on the total amount of saccharide or non-charged (neutral) oligosaccharide and its precursor(s) produced by said cell in the whole broth and / or supernatant, respectively. In a more preferred embodiment, the cell produces 25 g / L, 26 g / L, 1 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L, 75 g / L, 76 g / L, 77 g / L, 78 g / L, 79 g / L, 80 g / L, 81 g / L, 82 g / L, 83 g / L, 84 g / L, 85 g / L or more than 85 g / L of said saccharide or said non-charged (neutral) oligosaccharide in the whole broth and / or supernatant.

[0167] In another embodiment of present invention, the cell produces a saccharide as described herein.

[0168] In fourth aspect of present invention, the method for importing a disaccharide inside a cell according to the invention is used in a method for the production of a saccharide.

[0169] In a preferred embodiment of the method and / or cell of present invention, the cell produces a saccharide mixture comprising a saccharide as described herein. In a more preferred embodiment of the method and / or cell of present invention, the cell produces an oligosaccharide mixture comprising a saccharide as described herein

[0170] In another embodiment of present invention, the saccharide produced by a cell of present invention is separated and / or recovered from said cultivation or incubation medium and / or said cell. In a preferred embodiment, said saccharide is purified. The terms "separating from said cultivation or incubation medium and / or said cell" means harvesting, collecting, or retrieving said saccharide from the cell and / or the medium of its growth.

[0171] The saccharide can be separated in a conventional manner from the aqueous culture medium, in which the cell was grown. In case said saccharide is still present in the cells producing the saccharide, conventional manners to free or to extract said saccharide out of the cells can be used, such as cell destruction using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis,... The culture medium and / or cell extract together and separately can then be further used for separating said saccharide.

[0172] This preferably involves clarifying said saccharide to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing the genetically engineered cell. In this step, said saccharide can be clarified in a conventional manner. Preferably, said saccharide is clarified by centrifugation, flocculation, decantation and / or filtration. A second step of separating said saccharide preferably involves removing substantially all the eventually remaining proteins, peptides, amino acids, RNA, DNA, endotoxins and glycolipids that could interfere with the subsequent separation step, from said saccharide, preferably after it has been clarified. In this step, remaining proteins and related impurities can be removed from said saccharide in a conventional manner. Preferably, remaining proteins, salts, by-products, colour, endotoxins and other related impurities are removed from said saccharide by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, electrophoresis (e.g. using slab-polyacrylamide or sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including e.g. DEAE-sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices), ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and / or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. With the exception of size exclusion chromatography, remaining proteins and related impurities are retained by a chromatography medium or a selected membrane.

[0173] In a further preferred embodiment, the methods as described herein also provide for a further purification of the saccharide of present invention. A further purification of said saccharide be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange to remove any remaining DNA, protein, LPS, endotoxins, or other impurity. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Another purification step is accomplished by crystallization, evaporation or precipitation of said saccharide. Another purification step is to dry, e.g. spray dry or lyophilize the produced saccharide.

[0174] In an exemplary embodiment, the separation and purification of the saccharide is made in a process, comprising the following steps in any order: a) contacting the cultivation or a clarified version thereof with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the produced saccharide and allowing at least a part of the proteins, salts, by-products, colour and other related impurities to pass, b) conducting a diafiltration process on the retentate from step a), using said membrane, with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess of the electrolyte, c) and collecting the retentate enriched in said in the form of a salt from the cation of said electrolyte.

[0175] In an alternative exemplary embodiment, the separation and purification of said saccharide is made in a process, comprising the following steps in any order: subjecting the cultivation or a clarified version thereof to two membrane filtration steps using different membranes, wherein

[0176] - one membrane has a molecular weight cut-off of between about 300 to about 500 Dalton, and

[0177] - the other membrane as a molecular weight cut-off of between about 600 to about 800 Dalton.

[0178] In an alternative exemplary embodiment, the separation and purification of said saccharide is made in a process, comprising the following steps in any order comprising the step of treating the cultivation or a clarified version thereof with a strong cation exchange resin in H+-form and a weak anion exchange resin in free base form.

[0179] In an alternative exemplary embodiment, the separation and purification of said saccharide is made in the following way. The cultivation comprising the produced saccharide, biomass, medium components and contaminants, and wherein the purity of the produced saccharide in the cultivation is < 80 %, is applied to the following purification steps: i) separation of biomass from the cultivation, ii) cationic ion exchanger treatment for the removal of positively charged material, iii) anionic ion exchanger treatment for the removal of negatively charged material, iv) nanofiltration step and / or electrodialysis step, wherein a purified solution comprising the produced saccharide at a purity of greater than or equal to 80 % is provided. Optionally the purified solution is spray dried.

[0180] In an alternative exemplary embodiment, the separation and purification of the saccharide is made in a process, comprising the following steps in any order: enzymatic treatment of the cultivation; removal of the biomass from the cultivation; ultrafiltration; nanofiltration; and a column chromatography step. Preferably such column chromatography is a single column or a multiple column. Further preferably the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises i) at least 4 columns, wherein at least one column comprises a weak or strong cation exchange resin; and / or ii) four zones I, II, III and IV with different flow rates; and / or iii) an eluent comprising water; and / or iv) an operating temperature of 15 degrees to 60 degrees centigrade.

[0181] In a specific embodiment, the present invention provides the produced saccharide which is spray-dried to powder, wherein the spray-dried powder contains < 15 % -wt. of water, preferably < 10 % -wt. of water, more preferably < 7 % -wt. of water, most preferably < 5 % -wt. of water.

[0182] For identification of said saccharide as described herein, the monomeric building blocks (e.g. the monosaccharide or glycan unit composition), the anomeric configuration of side chains, the presence and location of substituent groups, degree of polymerization / molecular weight and the linkage pattern can be identified by standard methods known in the art, such as, e.g. methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography-mass spectrometry), MALDI-MS (Matrix-assisted laser desorption / ionization-mass spectrometry), ESI-MS (Electrospray ionization-mass spectrometry), HPLC (High-Performance Liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (High-Performance Anion-Exchange chromatography with Pulsed Amperometric Detection), CE (capillary electrophoresis), IR (infrared) / Raman spectroscopy, and NMR (Nuclear magnetic resonance) spectroscopy techniques. The crystal structure can be solved using, e.g., solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide-angle X-ray scattering). The degree of polymerization (DP), the DP distribution, and polydispersity can be determined by, e.g., viscosimetry and SEC (SEC-HPLC, high performance size-exclusion chromatography). To identify the monomeric components of the saccharide methods such as e.g. acid-catalysed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas-liquid chromatography) (after conversion to alditol acetates) may be used. To determine the glycosidic linkages, said saccharide is methylated with methyl iodide and strong base in DMSO, hydrolysis is performed, a reduction to partially methylated alditols is achieved, an acetylation to methylated alditol acetates is performed, and the analysis is carried out by GLC / MS (gas-liquid chromatography coupled with mass spectrometry). To determine the glycan sequence, a partial depolymerization is carried out using an acid or enzymes to determine the structures. To identify the anomeric configuration, said saccharide is subjected to enzymatic analysis, e.g., it is contacted with an enzyme that is specific for a particular type of linkage, e.g., beta-galactosidase, or alpha-glucosidase, etc., and NMR may be used to analyse the products. In another embodiment, the present invention provides use of a cell as described herein for the production of a saccharide as described herein.

[0183] In another embodiment, the present invention provides use of a method as described herein for the production of a saccharide as described herein.

[0184] In another embodiment, the present invention provides for a purified saccharide, or a purified oligosaccharide mixture comprising a saccharide as described herein for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.

[0185] In another embodiment, the present invention provides use of a purified saccharide obtained by a method as described herein in a food or feed preparation, in a dietary supplement, in a cosmetic ingredient or in a pharmaceutical ingredient. In some embodiments, said saccharide is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine. Said purified saccharide may be used for the manufacture of a preparation, as food additive, prebiotic, symbiotic, for the supplementation of baby food, adult food, infant animal feed, adult animal feed, or as either therapeutically or pharmaceutically active compound or in cosmetic applications. In another embodiment, the present invention provides use of milk oligosaccharide as described herein as additive in food, preferably as additive in human food and / or pet food, more preferably as additive in human baby food. In the context of present invention, the food is a human food, preferably infant food, human baby food and / or an infant formula or an infant supplement and the feed is a pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.

[0186] In another preferred embodiment, a preparation is provided that further comprises at least one probiotic microorganism. In another preferred embodiment of present invention, said preparation is a nutritional composition. In a more preferred embodiment, said preparation is a medicinal formulation, a dietary supplement, a dairy drink or an infant formula. A "prebiotic" is a substance that promotes growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract. In some embodiments, a dietary supplement provides multiple prebiotics, including said saccharide being a prebiotic purified by a method disclosed in this specification, to promote growth of one or more beneficial microorganisms. Examples of prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMOs) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharide). A "probiotic" product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (for example, L. acidophilus and L. bulgaricus), Bifidobacterium species (for example, B. animalis, B. longum and B. infantis (e.g., Bi-26)), and Saccharomyces boulardii. In some embodiments, said saccharide produced and / or purified by a method of this specification is orally administered in combination with such microorganism. Examples of further ingredients for dietary supplements include oligosaccharides (such as 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose), disaccharides (such as lactose), monosaccharides (such as glucose, galactose, L-fucose, sialic acid, glucosamine and N-acetylglucosamine), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skimmed milk, and flavourings.

[0187] In some embodiments, said saccharide purified by a method as described herein is incorporated into a human baby food (e.g., infant formula). Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk. In some embodiments, infant formula is sold as a powder and prepared for bottle- or cup-feeding to an infant by mixing with water. The composition of infant formula is typically designed to be roughly mimic human breast milk. In some embodiments, said saccharide purified by a method as described herein is included in infant formula to provide nutritional benefits similar to those provided by the oligosaccharides in human breast milk. In some embodiments, said purified saccharide is mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include non-fat milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and / or sunflower oil; and fish oils), vitamins (such as vitamins A, Bb, Bi2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMOs). In some embodiments, the one or more infant formula ingredients comprise non-fat milk, a carbohydrate source, a protein source, a fat source, and / or a vitamin and mineral. In some embodiments, the one or more infant formula ingredients comprise lactose, whey protein concentrate and / or high oleic safflower oil. In some embodiments, the concentration of the saccharide in the infant formula is approximately the same concentration as the concentration of the saccharide generally present in human breast milk. In some embodiments, a saccharide purified by a method as described herein is added to the infant formula with a concentration that is approximately the same concentration as the concentration of the compound generally present in human breast milk.

[0188] As will be shown in the examples herein, the methods and the cell of the invention preferably provide at least one of the following further surprising advantages as described herein:

[0189] - Higher titres of the saccharide (g / L) as described herein,

[0190] - Higher purity of the saccharide (g / L),

[0191] - A purity of the saccharide (g / L) equal to or greater than 80 %, preferably equal to or greater than 85 %, more preferably equal to or greater than 90 %, even more preferably equal to or greater than 91 %, even more preferably equal to or greater than 92 %, even more preferably equal to or greater than 93 %, even more preferably equal to or greater than 94 %, even more preferably equal to or greater than 95 %, even more preferably equal to or greater than 96 %, even more preferably equal to or greater than 97 %, even more preferably equal to or greater than 98 %, even more preferably equal to or greater than 99 %,

[0192] - Higher lactose conversion, leading to lower lactose concentration at end of fermentation (g / L lactose),

[0193] - Higher production rate r (g saccharide / L / h),

[0194] - Higher cell performance index CPI (g saccharide / g X),

[0195] - Higher specific productivity Qp (g saccharide / g X / h),

[0196] - Higher yield on the carbon source used Y (g saccharide / g carbon source used),

[0197] - Higher yield on sucrose Ys (g saccharide / g sucrose),

[0198] - Higher uptake / conversion rate of the carbon source used Q. (g carbon source / g X / h),

[0199] - Higher sucrose uptake / conversion rate Qs (g sucrose / g X / h),

[0200] - Higher lactose conversion / consumption rate rs (g lactose / h),

[0201] - Higher secretion, excretion or extracellular transport of the saccharide, and / or

[0202] - Higher growth speed of the production host, when compared to a method or a cell using an identical setup or enzymatic or genetic background but

[0203] - lacking introduction and expression and / or overexpression of a membrane protein as described herein, and / or

[0204] - (1) possessing and / or expressing at least gene encoding for a lactose permease and / or (2) possessing and / or expressing at least gene encoding for a lactose permease wherein said at least one gene encoding for a lactose permease has not been rendered less functional or has not been knocked-out as described herein.

[0205] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described above and below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are performed according to the manufacturer's specifications.

[0206] Further advantages follow from the specific embodiments and the examples. It goes without saying that the abovementioned features and the features which are still to be explained below can be used not only in the respectively specified combinations, but also in other combinations or on their own, without departing from the scope of the present invention. Moreover, the present invention relates to the following specific embodiments:

[0207] 1. A cell genetically engineered for the production of a non-charged (neutral) oligosaccharide, said cell comprising a pathway for production of said non-charged (neutral) oligosaccharide, wherein said cell is also genetically engineered to overexpress a membrane protein that (1) originates from the major facilitator superfamily (MFS) of transporters and that (2) is capable to transfer and / or transfers a disaccharide having a galactose (Gal) residue at its non-reducing end across the membrane of said cell, characterized in that said membrane protein comprises a polypeptide sequence comprising: the conserved domain CD-INB1 with sequence [AGSV][HKNQR][CDEGNQTV] wherein the second amino acid residue is aligned to Lys 18 in SEQ ID NO 01, and the conserved domain CD-INB2 with sequence [DEQ]XXX[FWY] wherein the first amino acid residue is aligned to Aspl24 in SEQ ID NO 01 and wherein X can be any amino acid residue.

[0208] 2. Cell according to embodiment 1, wherein said membrane protein:

[0209] (a) comprises a polypeptide sequence comprising: an IPR domain selected from the list consisting of or consisting essentially of IPR001927, IPR018043, IPR039672 and IPR036259 domain as defined by InterPro 98.0 as released on 25thJanuary 2024, the conserved domain cdl7332 domain as defined by the Conserved Domain Database CDD 3.20 as released on 6thOctober 2022, the PANTHER domain PTHR11328 as defined by PANTHER 18.0 as released on 17thSeptember 2023, and / or the PFAM domain PF13347 as defined by PFAM 32.0 as released on Sept 2018, and

[0210] (b) is any one of SEQ ID NOs 01, 02, 03, 04 or 05, a functional homolog or functional fragment of any one of said SEQ ID NOs 01, 02, 03, 04 or 05, a sequence having at least 40 % sequence identity to any one of the full-length amino acid sequences as represented by any one of SEQ ID NOs 01, 02, 03, 04 or 05, or a sequence having at least 40 %, at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by any one of SEQ ID NOs 01, 02, 03, 04 or 05.

[0211] 3. Cell according to any one of embodiment 1 or 2, wherein: the transfer activity of said membrane protein on said disaccharide is any one of i) influx, ii) efflux or iii) influx and efflux of said disaccharide across the membrane of said cell, and / or said membrane protein is capable to use and / or uses one or more coupling cation(s) for the transfer activity of said membrane protein on said disaccharide, wherein said coupling cation is selected from the list comprising, consisting of or consisting essentially of H+, Li+and Na+.

[0212] 4. Cell according to any one of previous embodiments, wherein said membrane protein is any one of an endogenous protein, a homologous protein or a heterologous protein of said cell.

[0213] 5. Cell according to any one of previous embodiments, wherein said cell comprises a polynucleotide sequence encoding said membrane protein and operably linked to control sequences recognized by the cell, said sequence (a) further i) being integrated in the genome of said cell and / or ii) presented to said cell on a vector and / or (b) being foreign to said cell.

[0214] 6. Cell according to any one of previous embodiments, wherein said pathway for production of said noncharged (neutral) oligosaccharide is selected from the list consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N- acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway.

[0215] 7. Cell according to any one of previous embodiments, wherein said cell further: comprises and / or is genetically engineered to comprise a pathway selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway, comprises at least one pathway selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway wherein at least one of said pathway(s) has / have been genetically engineered, possesses, expresses and / or overexpresses one or more glycosyltransferase(s) selected from the list consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N- acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N- acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl- beta-L-altrosamine transaminases, UDP- / V-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases, is capable to produce and / or produces one or more nucleotide-activated sugars, is genetically engineered for production of one or more of nucleotide-activated sugar(s), comprises a pathway for the synthesis of a nucleotide-activated sugar selected from the list consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N- acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2- acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N- acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP- Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-2-keto-3- deoxymanno-octulonic acid (CMP-KDO), GDP-rhamnose and UDP-xylose, possesses, expresses and / or overexpresses one or more genes selected from the list consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose- 1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L-glutamine— D-fructose-6- phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N- acetylmannosamine-6-phosphate 2-epimerase, UDP-GIcNAc 2-epimerase / kinase, glucosamine 6- phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase, Neu5Ac synthase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N- acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, d- arabinose 5-phosphate isomerase, KDO-8P synthase, KDO 8-phosphate phosphatase, CMP-KDO synthetase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, and / or comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and / or required for the synthesis of said non-charged (neutral) oligosaccharide.

[0216] 8. Cell according to any one of previous embodiments, wherein said non-charged (neutral) oligosaccharide is selected from the list consisting of or consisting essentially of non-charged (neutral) milk oligosaccharide; non-charged (neutral) mammalian milk oligosaccharide (MMO); non-charged (neutral) human milk oligosaccharide (HMO); fucosylated non-charged (neutral) oligosaccharide; non- fucosylated non-charged (neutral) oligosaccharide; fucosylated non-charged (neutral) milk oligosaccharide; non-fucosylated non-charged (neutral) milk oligosaccharide; fucosylated noncharged (neutral) MMO; non-fucosylated non-charged (neutral) MMO; fucosylated non-charged (neutral) HMO; non-fucosylated non-charged (neutral) HMO; N-acetylglucosamine containing noncharged (neutral) oligosaccharide; N-acetylglucosamine containing non-charged (neutral) milk oligosaccharide; N-acetylglucosamine containing non-charged (neutral) MMO; N-acetylglucosamine containing non-charged (neutral) HMO; N-acetyllactosamine containing non-charged (neutral) oligosaccharide; lacto-N-biose containing non-charged (neutral) oligosaccharide; galactosylated noncharged (neutral) oligosaccharide; galactosylated non-charged (neutral) milk oligosaccharide; an antigen of the human ABO blood group system; O-antigen; enterobacterial common antigen (ECA); an animal non-charged (neutral) oligosaccharide; a plant non-charged (neutral) oligosaccharide; chitosan; chitosan comprising non-charged (neutral) oligosaccharide; fucosylated non-charged (neutral) oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose ll7lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose; N-acetylglucosamine containing non-charged (neutral) oligosaccharide selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl- lacto-N-hexaose, difucosyl-lacto-N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH II), difucosyl- para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N- trifucoheptaose. Cell according to any one of previous embodiments, wherein said disaccharide having a Gal residue at its non-reducing end is selected from the list consisting of or consisting essentially of lactose, lactulose, melibiose, N-acetyllactosamine and lacto-N-biose. Cell according to any one of previous embodiments, wherein said cell: is capable to produce and / or produces said non-charged (neutral) oligosaccharide from one or more precursor(s), is capable to produce and / or produces said non-charged (neutral) oligosaccharide from lactose, is capable to produce and / or produces at least one precursor that is used to produce said non-charged (neutral) oligosaccharide, is capable to produce and / or produces all precursors that are used to produce said non- charged (neutral) oligosaccharide, is genetically engineered for the production of at least one precursor that is used to produce said non-charged (neutral) oligosaccharide, and / or is genetically engineered for the production of all precursors that are used to produce said non-charged (neutral) oligosaccharide. Cell according to embodiment 10, wherein at least one of said one or more precursor(s) is internalized in said cell via (1) one or more transporter protein(s) and / or (2) said membrane protein. Cell according to any one of previous embodiments, wherein said cell is: selected from the list consisting of prokaryotic cells and eukaryotic cells, optionally, said cell is selected from the list consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, fungal cells, plant cells, animal cells, insect cells, protozoan cells, a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell, preferably, said bacterium belongs to a phylum selected from the list comprising Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria; more preferably, said bacterium belongs to a family selected from the list comprising Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Corynebacteriaceae and Vibrionaceae; even more preferably, said bacterium is selected from the list comprising an Escherichia coli strain, a Bacillus subtilis strain, a Vibrio natriegens strain; even more preferably said Escherichia coli strain is a K-12 strain, most preferably said Escherichia coli K-12 strain is E. coli MG1655, preferably, said fungus belongs to a genus selected from the list comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, preferably, said yeast belongs to a genus selected from the list comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces, Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces or Torulaspora; more preferably, said yeast is selected from the list consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Yarrowia lipolytica, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii, preferably, said plant cell is an algal cell or is derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant, preferably, said animal cell is derived from insects, amphibians, reptiles, invertebrates, fish, birds or mammalian cells excluding human embryonic stem cells, more preferably said mammalian cell is selected from the list comprising an epithelial cell, an embryonic kidney cell, a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell, an NIH-3T3 cell, a lactocyte derived from mammalian induced pluripotent stem cells, more preferably said mammalian induced pluripotent stem cells are human induced pluripotent stem cells, a post-parturition mammary epithelium cell, a polarized mammary cell, more preferably said polarized mammary cell is selected from the list comprising live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells, live immortalized mammary epithelial cells, live immortalized mammary myoepithelial cells, live immortalized mammary progenitor cells, a non-mammary adult stem cell or derivatives thereof, more preferably said insect cell is derived from Spodoptera frugiperda, Bombyx mori, Mamestra brassicae, Trichoplusia ni or Drosophila melanogaster, preferably, said protozoan cell is a Leishmania tarentolae cell, an E. coll or yeast with a lactose permease positive phenotype, and / or an E. coll or yeast with a lactose permease positive phenotype wherein said lactose permease is coded by the gene LacY or LAC12, respectively. Method for the production of a non-charged (neutral) oligosaccharide, the method comprising the steps of: a) cultivating and / or incubating a cell, preferably a single cell, according to any one of embodiments 1 to 12 under conditions permissive to express said membrane protein and to produce said noncharged (neutral) oligosaccharide, b) preferably, separating, preferably purifying, said non-charged (neutral) oligosaccharide from the cultivation or incubation. Method according to embodiment 13, wherein the cultivation or incubation medium used in said cultivation or incubation, respectively, comprises one or more precursor(s) that is / are used for production of said non-charged (neutral) oligosaccharide. Method according to any one of embodiment 13 or 14, wherein at least one precursor feed for the production of said non-charged (neutral) oligosaccharide is added to said cultivation or incubation medium. Method according to any one of embodiments 13 to 15, the method comprising at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least one precursor; ii) Adding to the cultivation or incubation medium in a reactor or incubator at least one precursor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than twofold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed; iii) Adding to the cultivation or incubation medium in a reactor or incubator at least one precursor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than twofold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed and wherein preferably, the pH of said precursor feed is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feed is kept between 20°C and 80°C; iv) Adding at least one precursor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor feeding solution; v) Adding at least one precursor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor feeding solution and wherein the concentration of said precursor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of said precursor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feeding solution is kept between 20°C and 80°C; said method resulting in production of said non-charged (neutral) oligosaccharide with a concentration of at least 25 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 75 g / L, at least 90 g / L, at least 100 g / L, at least 125 g / L, at least 150 g / L, at least 175 g / L and / or at least 200 g / L in the final volume of the cultivation or incubation.

[0217] 17. Method according to any one of embodiments 13 to 16, the method comprising at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter); ii) Adding to the cultivation or incubation medium in a reactor or incubator a precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two- fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed; iii) Adding to the cultivation or incubation medium a precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed and wherein preferably, the pH of said precursor feed is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feed is kept between 20°C and 80°C; iv) Adding a precursor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor feeding solution and wherein the concentration of said precursor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably the pH of said precursor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feeding solution is kept between 20°C and 80°C; said method resulting in production of said non-charged (neutral) oligosaccharide with a concentration of at least 25 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 75 g / L, at least 90 g / L, at least 100 g / L, at least 125 g / L, at least 150 g / L, at least 175 g / L and / or at least 200 g / L in the final volume of the cultivation or incubation. Method according to any one of embodiments 13 to 17, wherein said precursor is selected from the list comprising, consisting of or consisting essentially of glucose, galactose, GIcNAc, GalNAc, UDP- GIcNAc, UDP-GalNAc, UDP-Gal, lactose, GlcNAc-pi,3-Gal-pi,4-Glc (LN3, lacto-N-triose), Gal-pi,3- GlcNAc-pi,3-Gal-pi,4-Glc (LNT, lacto-N-tetraose) and Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc (LNnT, lacto-N-neotetraose). Method according to any one of embodiments 13 to 18, wherein said cultivation or incubation medium contains at least one: compound selected from the list consisting of or consisting essentially of lactose, galactose, glucose, UDP-Gal, UDP-GIcNAc and UDP-GalNAc, carbon source selected from the list comprising, consisting of or consisting essentially of a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract; and / or carbon source selected from the list comprising, consisting of or consisting essentially of glucose, N-acetylglucosamine (GIcNAc), glycerol, fructose, sucrose, maltose, lactose, arabinose, maltooligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate. Method according to any one of embodiments 13 to 19, wherein said cell produces 25 g / L or more of said non-charged (neutral) oligosaccharide in the whole broth and / or supernatant and / or wherein said non-charged (neutral) oligosaccharide in the whole broth and / or supernatant has a purity of at least 80 % measured on the total amount of non-charged (neutral) oligosaccharide and its precursor(s) produced by said cell in the whole broth and / or supernatant, respectively. Method according to any of embodiments 13 to 20, wherein said separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and / or gel filtration, ligand exchange chromatography. Method according to any one of embodiments 13 to 21, wherein said purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization. Use of a cell according to any one of embodiments 1 to 12 for the production of a non-charged (neutral) oligosaccharide, wherein said non-charged (neutral) oligosaccharide is selected from the list consisting of or consisting essentially of non-charged (neutral) milk oligosaccharide; non-charged (neutral) mammalian milk oligosaccharide (MMO); non-charged (neutral) human milk oligosaccharide (HMO); fucosylated non-charged (neutral) oligosaccharide; non-fucosylated non-charged (neutral) oligosaccharide; fucosylated non-charged (neutral) milk oligosaccharide; non-fucosylated noncharged (neutral) milk oligosaccharide; fucosylated non-charged (neutral) MMO; non-fucosylated non-charged (neutral) MMO; fucosylated non-charged (neutral) HMO; non-fucosylated non-charged (neutral) HMO; N-acetylglucosamine containing non-charged (neutral) oligosaccharide; N- acetylglucosamine containing non-charged (neutral) milk oligosaccharide; N-acetylglucosamine containing non-charged (neutral) MMO; N-acetylglucosamine containing non-charged (neutral) HMO; N-acetyllactosamine containing non-charged (neutral) oligosaccharide; lacto-N-biose containing noncharged (neutral) oligosaccharide; galactosylated non-charged (neutral) oligosaccharide; galactosylated non-charged (neutral) milk oligosaccharide; an antigen of the human ABO blood group system; O-antigen; enterobacterial common antigen (ECA); an animal non-charged (neutral) oligosaccharide; a plant non-charged (neutral) oligosaccharide; chitosan; chitosan comprising noncharged (neutral) oligosaccharide; fucosylated non-charged (neutral) oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6- fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose ll7lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N- hexaose and difucosyl-lacto-N-neohexaose; N-acetylglucosamine containing non-charged (neutral) oligosaccharide selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto- N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N- neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N-hexaose, difucosyl- lacto-N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH II), difucosyl-para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N-trifucoheptaose. Use of a method according to any one of embodiments 13 to 22 for the production of a non-charged (neutral) oligosaccharide, wherein said non-charged (neutral) oligosaccharide is selected from the list consisting of or consisting essentially of non-charged (neutral) milk oligosaccharide; non-charged (neutral) mammalian milk oligosaccharide (MMO); non-charged (neutral) human milk oligosaccharide (HMO); fucosylated non-charged (neutral) oligosaccharide; non-fucosylated non-charged (neutral) oligosaccharide; fucosylated non-charged (neutral) milk oligosaccharide; non-fucosylated noncharged (neutral) milk oligosaccharide; fucosylated non-charged (neutral) MMO; non-fucosylated non-charged (neutral) MMO; fucosylated non-charged (neutral) HMO; non-fucosylated non-charged (neutral) HMO; N-acetylglucosamine containing non-charged (neutral) oligosaccharide; N- acetylglucosamine containing non-charged (neutral) milk oligosaccharide; N-acetylglucosamine containing non-charged (neutral) MMO; N-acetylglucosamine containing non-charged (neutral) HMO; N-acetyllactosamine containing non-charged (neutral) oligosaccharide; lacto-N-biose containing noncharged (neutral) oligosaccharide; galactosylated non-charged (neutral) oligosaccharide; galactosylated non-charged (neutral) milk oligosaccharide; an antigen of the human ABO blood group system; O-antigen; enterobacterial common antigen (ECA); an animal non-charged (neutral) oligosaccharide; a plant non-charged (neutral) oligosaccharide; chitosan; chitosan comprising noncharged (neutral) oligosaccharide; fucosylated non-charged (neutral) oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6- fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N- hexaose and difucosyl-lacto-N-neohexaose; N-acetylglucosamine containing non-charged (neutral) oligosaccharide selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto- N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N- neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N-hexaose, difucosyl- lacto-N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH ll)7difucosyl-para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N-trifucoheptaose.

[0218] The invention will be described in more detail in the examples. The following examples will serve as further illustration and clarification of the present invention and are not intended to be limiting.

[0219] Examples

[0220] Example 1. Calculation of percentage identity between nucleotide or polypeptide sequences

[0221] Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. (1970) 48: 443-453) to find the global (i.e., spanning the full-length sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al., J. Mol. Biol. (1990) 215: 403-10) calculates the global percentage sequence identity (i.e., over the full-length sequence) and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologs may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity (i.e., spanning the full-length sequences) may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics (2003) 4:29). Minor manual editing may be performed to optimize alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologs, specific domains may also be used, to determine the so-called local sequence identity. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence (= local sequence identity search over the full-length sequence resulting in a global sequence identity score) or over selected domains or conserved motif(s) (= local sequence identity search over a partial sequence resulting in a local sequence identity score), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7).

[0222] Example 2. Identification of a membrane protein of present invention

[0223] MFS transporters can be obtained from sequence databases like Uniprot (https: / / www.uniprot.org / ), NCBI nr or nt databases (https: / / www.ncbi.nlm.nih.gov / ) and others. This example describes how to extract IPR001927 transporters comprising a polypeptide sequence comprising a lactose binding pocket. Members of IPR001927 were extracted using the Uniprot sequence database. In total 21226 protein sequences were found (09 / 11 / 2023). Sequences were aligned with the polypeptide with SEQ ID NO 01 using clustalomega (https: / / www.ebi.ac.uk / Tools / msa / clustalo) and were filtered for sequences comprising (i) the conserved domain CD-INB1 with sequence [AGSV][HKNQR][CDEGNQTV] wherein the second amino acid residue is aligned to Lys 18 in SEQ ID NO 01 and (ii) the conserved domain CD-INB2 with sequence [DEQ]XXX[FWY] wherein the first amino acid residue is aligned to Aspl24 in SEQ ID NO 01 and wherein X can be any amino acid residue. Amino acid sequences were clustered using CD-HIT (http: / / weizhongli-lab.org / cd-hit / ) with a sequence identity threshold of 80% and further filtered on completeness and source.

[0224] The representative sequences are the next 600 identifiers: A0A847PIZ8, A0A7C6BET9, A0A7X1HRK7, A0A7X1U30, A0A9E2KVR2, A0A9E2KXR9, A0A943JT22, A0A943B048, A0A6N7VHN1, A0A431ISA9, A0A0T6TYA0, A0A1N6NSB4, A0A9E2KMT2, A0A948TEI5, A0A948X0L0, A0A9D1WDV8, E8LHF5, A0A9D9GT73, A0A3D1YJK6, A0A2E6XLE4, A0A4R6DWM5, A0A7Y7YK91, A0A5Q2K7G0, A0A837JSU2, A0A7D6FGY7, A0A379THH5, A0A0L7TDF7, A0A2K1Q4J2, A0A2K1QB22, A0A9Q6DJG5, H3RH71, A0A0J8YMS1, A0A0N0IAH6, A0A4R3VV75, A0A2I0G0R1, A0A6M2B0J9, A0A542BKC8, A0A7Y8RHN7, A0A318UZU2, F6CWF5, A0A4V0IEB6, A0A356E9E1, A0A946P6G5, A0A1B9PFV1, A0A855S8J6, A0A2T3HSG3, A0A2T3J2Q2, Q1ZA37, Q1YYS0, A0A7Y4F282, A0A1B1NVS5, A0A7Y4C4N0, A0A0W8JDP0, A0A7Y4DC45, A0A1B1ELE7, A0A431I1F4, F0RS83, G8QSG5, G8QRP4, A0A3C1LRM6, A0A7X2TQV9, A0A841RF08, A0A841RAJ2, A0A841R8N9, A0A1I3HUA5, A0A9Q9EWU1, A0A7S6WM73, A0A7H8VKB7, A0A7W8LLT0, A0A3C0BHX3, A0A352TCC8, A0A7C6MB68, A0A847MYA9, A0A971H402, A0A970RP56, A0A7X9IRE9, A0A832KNJ0, A0A2G6HZA1, A0A2N1TIA1, A0A2N1SNE9, A0A2N1SHP5, A0A2N1RYE7, A0A2N1RIC1, A0A1V5WJ56, A0A1G3MSN4, A0A947QEF9, A0A7V3Y2N8, A0A947QGS0, A0A1Q5PZQ5, A0A7K0K7F9, A0A975XXW1, A0A9E1C6D6, A0A7X7CB11, A0A261G7A6, A0A430FBH6, A0A6I5N1T5, A0A1C4H4W0, A0A2M9H8U2, A0A0M4M2L4, A0A7Y0HTP0, W6K2B6, A0A077M2K6, A0A936USR6, A0A191WB19, A0A4S4FWI6, A0A1G7UV60, A0A4V3WTG0, A0A387B7R3, A0A5C1Y4H8, A0A7G9XRX0, A0A7G9XPP6, A0A8J3GRX2, A0A3E0VKD0, A0A4V2EYA2, A0A7M4DPX4, A0A9D2J4E0, A0A239QWC5, A0A7W5FGS9, A0A327Z072, A0A810NR80, A0A8J3HG88, A0A2N9JC79, A0A2A9CQW2, K7RKW1, A0A4P8ZS03, A0A3P1WQC7, A0A255ENF6, A0A7C6IDN0, A0A6G7YAT9, A0A4Q9KJ96, A0A4Q9KMB3, A0A5Q2F8T7, A0A7H0H7I1, A0A1Q2CUF9, A0A9D7P9R0, A0A3P1WHC6, A0A1R1IKF7, A0A3C1FB48, A0A2N3GGB2, A0A2N3FWI8, A0A949DYF0, A0A9E1F6L0, A0A9E1BKA4, A0A947R7F6, A0A9D1ZXZ4, A0A970D9T4, A0A970DBM8, A0A972ECN2, A0A972EE24, A0A9E2GAE3, A0A970ALB4, A0A3C0DIK3, A0A971LBL3, A0A7C6GM62, A0A2V2GHD1, A0A350X468, A0A357NEQ7, A0A940ZRF7, A0A940ZTW6, A0A9E1CDE5, A0A350WZ19, A0A356BZH3, A0A640MMR1, A0A2B2F0G7, A0A2A7WQ92, A0A2A8SME2, A0A1G9WJI5, A0A1W6A0Q8, A0A150KC89, A0A1M5LHP4, A0A0A2UU02, A0A2C5HM02, A0A3N6B4U8, A0A2A9Y0R8, A0A1H9U9I6, A0A919XRI3, A0A1R0YCE6, A0A920CVE1, A0A3D9SJ21, A0A1I6VRZ8, A0A1R0ZVT2, A0A1H7ZD46, A0A810DW76, A0A4Y9K8N7, A0A9D1QFF5, A0A380BVZ4, A0A143PCQ0, A0A9Q6HR39,S2XG40, A0A6P1WYN9, A0A6P1WSY1, A0A7X8C498, A0A8B5W4G1, A0A143YKL8, A0A6B1T7X8, S1MTC0, Q3Y292, R2SGF4, R2Q8P3, S0L2K9, A0A242CYF9, X0PNS7, A0A347STM8, A0A0H4QGR7, A0A6N9I4Y2, A0A0R2L4K3, A0A5P8JMF7, A0A9N7EHW7, A0A9D9H7Y9, A0A4V3REA8, A0A7L5UK50, A0A7L5UFF5, N2A0T1, N1ZU84, A0A8E2X9U6, A0A8E1X3F7, A0A401FJ64, A0A401FJ76, A0A0R1SES8, A0A0R1W0X1, A0A0R1VRP7, A0A0R1YTE7, A0A0R1YJL0, A0A5A5U1C6, A0A5B8TDT6, A0A5B8T1H0, D5T091, C2KJ64, A0A7Z6QIQ5, A0A921EFN1, A0A921EG71, A0A0F3RS37, A0A0R1LEV3, A0A0R1GT62, A0A0R1GQ84, A0A0R2DJJ2, A0A9Q9J787, A0A6F9Y489, A0A062X9L8, A0A179CKK3, A0A837IWL5, A0A089QAJ1, A0A0R1TS06, A0A7W3TSD5, A0A839H7I9, A0A1L7GVD6, A0A099YA98, A0A363EAK3, C7XWC4, A0A0R1UVT1, A0A0R1P5L6, A0A0R1VAD5, A0A0R1V755, A0A0R2E3U5, A0A5Q2P4S9, A0A5B8TG66,Q04GZl, A0A0D0Y4M5, A0A0R1RL35, A0A1Y0WGD1, A0A0R1N4T0, A0A1Z5I8K3, A0A1Z5IAE6, A0A1B2IUU3, A0A1Z5IV74, A0A1Z5IZ32, A0A0R1TEQ0, A0A0R1TK10, A0A1X4JL40, A0A5M9EJI6, A0A288Q779, A0A069D2T3, A0A7L4W7N7, A0A6L2ZYF5, A0A6A0B8P8, A0A2A5RPM5, A0A0V8D3X3, A0A0V8DNM7, A0A7G2N3D9, A0A1G6A600, A0A2I1TLQ7, A0A7X7Q3F9, A0A7X7Q369, A0A9D1IFM7, A0A9D1IFA8, AOA9D1IEP7,F2JQN2, A0A7X8D8T4, A0A252F627, A0A174R7J1, A0A7G5P0W7, A0A174HT13, A0A174FHK4, A0A5N7INB0, A0A2A7MBG7, W1UJV8, W1UB75, A0A1C5VA88, A0A1C6K802, A0A1C5QVC3, A0A1C6HZ72, A0A1C5PXG5, A0A1C5M6N6, A0A1C5Q8V8, A0A943XJ33, A0A943S6Z9, A0A7X7B4A3, A0A7X6TQC8, A0A3D5MWL5, A0A943BB97, A0A943BAN9, A0A939KKH6, A0A1I5EX67, A0A173ZR26, A0A1G9L774, A0A7X8JIR0, A0A7X6YQM7, A0A357T9P1, A0A970IU02, A0A970RV86, A0A7X7KSQ9, A0A7X8JDY9, A0A7X6Y2X7, A0A970A407, A0A971JFE8, A0A7X8ZLD2, A0A972IBN9, A0A971FT07, A0A7C6BZR4, A0A971F382,V7ll80, A0A1I7FD43, A0A173VCM3, A0A1T4PZD3, A0A350VX46, R6QA58, R6Q979, A0A9D1G302, A0A9D1MIA9, A0A9D1NB54, A0A9D1SX65, A0A9D1NCA8, A0A9D1SX50, A0A9D1NBR0, A0A9D1NCS8, A0A940DGD5, A0A940DF62, A0A349G8D6, A0A349G700, A0A1Y4TMN8, A0A7X2MCK4, A0A5S4VU04, A0A1I5D7Z2, A0A6P1TIK1, A0A7R7EJZ8, AOA1I6INCO,BOMBI2, A0A7X2TNJ8, A0A4R2JB87, A0A1H9LDH7, A0A9D5MI78, A0A849XV00, A0A6N9K711, A0A850HI32, R6WMG2, A0A9D2C702, A0A6N7WHY2, A0A6N7VWL2, A0A6M5G193, A0A174Q.J07, A0A2Y9BAW4, A0A0H5SH31, A0A1H3H060, A9KHL8, A0A1H5RT86, A0A174HXS3, A0A1G5RZE4, A0A0J1G9Y4, A0A0M6WLD5, A0A6L6L7S6, A0A174BN59, A0A6L5YSF3, G2T1H3, A0A3D2PUB2, A0A1Q9J590, C4G992, A0A7X3MM69, A0A1Y4UDM4, A0A1Y4UGU1, A0A7U9QZ42, A0A3D0MAZ3, A0A348N3Z4, A0A927YPG9, A0A927YG50, A0A3B9IAA0, A0A3D5L010, A0A349BZJ0, A0A927T641, A0A316Q758, A0A927TML5, A0A9D5MTM9, A0A927VMA4, A0A7U9SH75,R9MR72, A0A1H6RIU8, A0A1G4RY43, A0A1G5VMH4, A0A7U6QKX6, R9JWA0, A0A1H9F4C6, A0A1H9E892, A0A1G5GIF3, A0A1I6X1F3, A0A1I6X776, A0A1G4VFG4, A0A1G4VUN1, A0A839JZQ7, A0A839K2Q7, A0A1H0BYD7, A0A9E1I3U1, A0A844DWZ4, R5FGX0, R5FI43, A0A1C7FQH1, A0A9D1V326, A0A9D1V4T0, A0A9D1MV45, A0A9D1MVC6, A0A9D1SMN9, A0A9D5R7B5, A0A4P8XWQ9, A0A943PJ01, A0A943S2G8, R7FEZ8, R5XXU5, R5YVB9, A0A0D8IY45, A0A928JQ48, A0A355G430, A0A355G3C5, A0A9D5YD25, A0A3D5TR77, A0A9D5TA92, A0A3D2TMX2, A0A928MJG3, A0A928PUV2, A0A928PSR4, A0A928PLZ9, A0A928PS37, A0A928HYV8, A0A928JP05, A0A940ZLW4, A0A847PMD4, A0A9D5SWR1, A0A9D5YAM1, A0A847C499, A0A928M0R5, A0A942Y683, A0A943AQC0, A0A1M6K6R5, A0A942J5E6, A0A970RRS6, A0A349Q7X7, A0A7C6TNC2, A0A3D2G0J9, A0A943Q645, A0A970UM73, A0A970UKW6, A0A927ZGD0, A0A316RBM9, A0A943KDB4, A0A970RQA1, A0A927W1J7, A0A943K8B6, A0A316R9K7, A0A3C0NME3, A0A971WCS4, A0A7C6NXV0, A0A7C7ACN9, A0A970EW80, A0A971G1C1, A0A7C6BG40, A0A970G4S7, A0A970VGU1, A0A972CWU6, A0A7C6DDD0, A0A927SLW9, A0A3D4QY31, A0A2V2DR69, A0A3P2ALM3, A0A9D9T3E6, A0A7X7N0D6, A0A9D9WLF4, A0A2V2FSD1, A0A101WC77, A0A6N9L9E8, A0A1H2UNF5, A0A844FX84, A0A9Q2X2T0, A0A7C6SH74, A0A7X7AD97, A0A3G9JE08, A0A7X2NR63, A0A927XDA4, A0A927X3U5, A0A7X8H707, A0A847LSG8, A0A3B9G122, A0A3B9G2R0, A0A3D2W8W3, A0A9D5NB54, A0A4S2F8U5, A0A9E6F8R2, A0A357R2I6, A0A3D4VPX6, A0A1C0BXZ7, A0A9Q9CHJ9, A0A173QWR4, A0A848Q8Q9, A0A970SSD9, A0A942KFU8, R7KAA3, R7KAU9, A0A6I2UET4, A0A9E1HSS1, A0A9E1HRB2, A0A239U024, A0A4V6NYX4, A0A1H6Z0D9, A0A928A0L9, A0A930M443,C7RF64, A0A1V6FMC8, A0A1V6FK98, A0A1V6FPG8, A0A1V6FK47, A0A1V6FRM9, A0A1V6AIR0, A0A1V5YDH5, A0A1V5Y7X2, A0A1V5U460, A0A1V5TLZ3, A0A1V5TMW2, A0A1V5TSD7, A0A1V5TM R7, A0A1V5S6E0, A0A1V5S2E6, A0A1V5S2T2, R5EK26, R6WDK4, R6I5K1, R7BE84, R7N471, A0A1F9ZSC8, A0A2N2DXT3, A0A2N2CW69, A0A2N2A5P1, A0A419ECE9, A0A1B3WU18, A0A449BIZ5, A0A7C6HJ37, A0A3A6NP42, U4KN09, A0A7X7LSZ5, A0A970WZD3, A0A7C6Y5F3, A0A7X7UQ99, A0A970IW65, A0A7C6SCF7, A0A7V6GNJ2, A0A651DLC7, A0A7V6IV23, A0A7X9A4V7, A0A348NKC5, A0A356L4V4, A0A1V6BNI6, A0A1V5HNZ9, A0A1G3W425, A0A2N1QIT3, A0A2N1QCQ8, A0A2N1Q535, A0A2N1PYF2, A0A2K9AIR8, A0A1G3X6E5, A0A7C6M3P4, A0A644XJY2, A0A0L0RIA8, A0A5M9DJF2, A0A0N8VY07, A0A6I6IB44, A0A1L3KBK7, A0A2N6TXC7, A0A1E5TRW5, A0A5M9DHZ3, A0A5B8TTT4, A0A558LSN9, A0A929W8Q4,

[0225] A0AlE5TF10,J8AVR3, A0A7Y4BND0.

[0226] Example 3. Materials and Methods

[0227] A. Escherichia coli Media

[0228] The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The minimal medium used in the cultivation experiments in 96-well plates or in shake flasks contained 2.00 g / L NH4CI, 5.00 g / L (NH4)2SO4, 2.993 g / L KH2PO4, 7.315 g / L K2HPO4, 8.372 g / L MOPS, 0.5 g / L NaCI, 0.5 g / L MgSO4.7H2O, 30 g / L sucrose or 30 g / L glycerol, 1 ml / L vitamin solution, 100 pl / L molybdate solution, and 1 mL / L selenium solution. As specified in the respective examples, for the production of a saccharide, 0.30 g / L sialic acid, 0.30 g / L GIcNAc, 20 g / L lactose, 20 g / L 2'FL, 20 g / L 3-FL, 20 g / L DiFL, 20 g / L LN3, 20 g / L LNT, 20 g / L LNnT, 20 g / L LacNAc and / or 20 g / L LNB was additionally added to the medium. The minimal medium was set to a pH of 7.0 with IM KOH. Vitamin solution consisted of 3.6 g / L FeCI2.4H2O, 5.0 g / L CaCI2.2H2O, 1.3 g / L MnCI2.2H2O, 0.38 g / L CuCI2.2H2O, 0.5 g / L CoCI2.6H2O, 0.94 g / L ZnCI2, 0.0311 g / L H3BO4, 0.4 g / L Na2EDTA.2HzO and 1.01 g / L thiamine. HCI. The molybdate solution contained 0.967 g / L NaMoO4.2H2O.

[0229] The selenium solution contained 42 g / L Seo2.

[0230] The minimal medium for fermentations contained 6.75 g / L NH4CI, 1.25 g / L (NF hSC , 2.93 g / L KF PC and 7.31 g / L KH2PO4, 0.5 g / L NaCI, 0.5 g / L MgSO4.7H2O, 30 g / L sucrose or 30 g / L glycerol, 1 mL / L vitamin solution, 100 p.L / L molybdate solution, and 1 mL / L selenium solution with the same composition as described above. As specified in the respective examples, for the production of a saccharide, 0.30 g / L sialic acid, 0.30 g / L GIcNAc, 20 g / L lactose, 20 g / L 2'FL, 20 g / L 3-FL, 20 g / L Di FL, 20 g / L LN3, 20 g / L LNT, 20 g / L LNnT, 20 g / L LacNAc and / or 20 g / L LNB was additionally added to the medium. Complex medium was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic: e.g., chloramphenicol (20 mg / L), carbenicillin (100 mg / L), spectinomycin (40 mg / L) and / or kanamycin (50 mg / L). When necessary, an inducer for inducible gene expression like e.g., IPTG or arabinose was added.

[0231] Plasmids pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT-flanked kanamycin resistance (kan) gene), and pCP20 (expresses FLP recombinase activity) plasmids were obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium in 2007). The pET28b(+) vector was obtained from Millipore and adapted for Golden Gate cloning. Plasmids were maintained in the host E. coli DH5alpha (F", phi80d / ocZZ!M15, t (lacZYA-argF) U169, deoR, recAl, endAl, hsdR17(rk", mk+), phoA, supE44, lambda", thi-1, gyrA96, relAl) bought from Invitrogen.

[0232] Strains and mutations

[0233] Escherichia coli K12 MG1655 [X", F", rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain#: 7740, in March 2007. E. coli NiCo21 (DE3) was obtained from New England Biolabs, in November 2017. E. coli SHuffle T7 Express was obtained from New England Biolabs, in December 2021. E. coli Origami 2 (DE3) was obtained from Merck Life Science (Sigma), in December 2021. Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645) as described in e.g., WO22034067. Gene mutations were created via a PCR-based method described by Sanchis et al. (Appl. Microbiol. Biotechnol. (2008) 81(2), 387-397). In an example to produce LN3, the mutant strain was derived from E. coli K12 MG1655 and modified with a knock-out of the E. coli LacZ and nagB genes and with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid like e.g. a pSClOl-derived plasmid, for a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA with UniProt ID Q9JXQ6 from Neisseria meningitidis. In some examples for the production of a saccharide the mutant strain was additionally modified with the knock-out of the E. coli LacY gene. In an example for production of LN3 derived oligosaccharides like lacto- / V-tetraose (LNT, Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc), the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1, 3- galactosyltransferase like e.g. wbgO (Uniprot ID D3QY14) from E. coli 055:1-17. In an example for production of LN3 derived oligosaccharides like lacto- / V-neotetraose (LNnT, Gal-bl,4-GlcNAc-bl,3-Gal- bl,4-Glc), the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N- acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis.

[0234] Optionally, the LN3, LNT and / or LNnT production is further optimized in the mutant E. coli strains with a genomic knock-in of one or more constitutive transcriptional units for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, LN3, LNT and / or LNnT production may be further optimized in the mutant E. coli strains by overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. LN3, LNT and / or LNnT production can further be optimized in the mutant E. coli strains with genomic knock-outs of the E. coli genes comprising any one or more of galT, ushA, IdhA and agp.

[0235] The mutant LN3, LNT and / or LNnT producing strains can also be optionally modified for enhanced UDP- GIcNAc production with a genomic knock-in of a constitutive transcriptional unit for an L-glutamine— D- fructose-6-phosphate aminotransferase like e.g. the mutant glmS*54 from E. coli (differing from the wildtype E. coli glmS protein, having UniProt ID P17169 (sequence version 04, 23 Jan 2007), by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 2006, 88: 419-429). The mutant E. coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g. galE from E. coli (UniProt ID P09147), a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03, 23 Jan 2007) and an N- acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7). The mutant LN3, LNT and / or LNnT producing E. coli strains can also optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6). Alternatively, and / or additionally, production of LN3, LNT, LNnT and oligosaccharides derived thereof can further be optimized in the mutant E. coli strains with genomic knock-ins of constitutive transcriptional units comprising a membrane transporter protein like e.g. MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID P0AEY8) and MdfA from Yokenella regensburgei (UniProt ID G9Z5F4).

[0236] In an example for GDP-fucose production, the mutant derived from E. coli K12 MG1655 as described e.g. in WO22034067, WO220234068 or WO22034069. In a next step, the mutant strain was further modified with a genomic knock-out of the E. coli LacY gene. To allow fucosylated oligosaccharide production, the mutant E. coli strain producing GDP-fucose was further modified with one or more transcriptional unit(s) encoding one or more fucosyltransferases. Optionally, the strain is additionally modified with a genomic knock-in of one or more constitutive transcriptional units for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re- introduction of an expression module at euchromatin, usage of high-copy-number plasmids. The mutant E. coli strain can also optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6).

[0237] In an example to produce one or more fucosylated non-charged oligosaccharide(s), an E. coli K12 M1655 strain is modified for production of GDP-fucose, LN3, LNT and / or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, an E. coli K12 MG1655 strain is modified for production of CMP-sialic acid, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, an E. coli K12 MG1655 strain is modified for production of CMP-sialic acid, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).

[0238] Preferably but not necessarily, any one or more of the glycosyltransferases and / or the proteins involved in nucleotide-activated sugar synthesis were N- and / or C-terminally fused to a solubility enhancer tag like e.g. a SUMO-tag, an MBP-tag, His, FLAG, Strep-ll, Halo-tag, NusA, thioredoxin, GST and / or the Fh8-tag to enhance their solubility. Optionally, the modified E. coli strains were modified with a genomic knock-ins of a constitutive transcriptional unit encoding a chaperone protein like e.g. DnaK, DnaJ, GrpE or the GroEL / ES chaperonin system. All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139 5148), Dunn et al. (Nucleic Acids Res. 1980, 8, 2119-2132), Edens et al. (Nucleic Acids Res. 1975, 2, 1811-1820), Kim and Lee (FEBS Letters 1997, 407, 353-356) and Mutalik et al. (Nat. Methods 2013, No. 10, 354-360). All strains were stored in cryovials at -80°C (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).

[0239] In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from E. coli K12 MG1655 as described e.g. in WO22034067, WO22034068 or W022034070. In a next step, the mutant strain was further modified with a genomic knock-out of the E. coli LacY gene. To allow sialylated oligosaccharide production, the mutant E. coli strain producing CMP-sialic acid was further modified with one or more transcriptional unit(s) encoding one or more sialyltransferases. Optionally, the strain is additionally modified with a genomic knock-in of one or more constitutive transcriptional units for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. The mutant E. coli strain can also optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6).

[0240] Cultivation conditions

[0241] A preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 pL LB and was incubated overnight at 37 °C on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96well square microtiter plate, with 400 pL minimal medium by diluting 400x. These final 96-well culture plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72h, or shorter, or longer. To measure sugar concentrations at the end of the cultivation experiment whole broth samples were taken from each well by boiling the culture broth for 1 hour at 60°C before spinning down the cells (= average of intra- and extracellular sugar concentrations).

[0242] A preculture for the bioreactor was started from an 250pl cryovial of a certain strain, inoculated in 250 mL or 500 mL minimal medium in a 1 L or 2.5 L shake flask and incubated for 24 h at 37°C on an orbital shaker at 200 rpm. A 5 L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culturing condition were set to 37 °C, and maximal stirring; pressure gas flow rates were dependent on the strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H2S04 and 20% NH4OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.

[0243] B. Saccharomyces cerevisiae

[0244] Media

[0245] Strains were grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) containing 6.7 g / L Yeast Nitrogen Base without amino acids (YNB w / o AA, Difco), 20 g / L agar (Difco) (solid cultures), 22 g / L glucose monohydrate, 0.79 g / L CSM or 0.77 g / L CSM-Ura, 0.77 g / L CSM-Trp, or 0.77 g / L CSM-His (MP Biomedicals). For the production of a saccharide, also, 0.30 g / L sialic acid, 0.30 g / L GIcNAc, 20 g / L lactose, 20 g / L 2'FL, 20 g / L 3-FL, 20 g / L DiFL, 20 g / L LN3, 20 g / L LNT, 20 g / L LNnT, 20 g / L LacNAc and / or 20 g / L LNB may be added to the medium.

[0246] Strains

[0247] S. cerevisiae BY4742 created by Brachmann et al. (Yeast (1998) 14:115-32) was used, available in the Euroscarf culture collection. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355-360, 1995).

[0248] In an example to produce UDP-galactose, a yeast expression plasmid derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the HIS3 selection marker was modified as described e.g., in WO22034067. In an example to produce LN3, said plasmid was further modified with transcriptional units encoding (1) a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 and (2) a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (UniProt ID Q9JXQ6). Alternatively, and / or additionally, the mutant yeast strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re- introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In another example, the S. cerevisiae strain engineered for LN3 production is further modified with a transcriptional unit for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli O55:H7 to produce LNT or with an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis to produce LNnT.

[0249] In an example to produce GDP-fucose, the yeast expression plasmid p2a_2p_Fuc (Chan 2013, Plasmid 70, 2-17) comprising an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli and the 2p yeast ori and the Ura3 selection marker for selection and maintenance in yeast was modified as described in e.g., WQ22034067. To allow fucosylated oligosaccharide production, the mutant S. cerevisiae strain producing CMP-sialic acid was further modified with one or more transcriptional unit(s) encoding one or more fucosyltransferases. In a next step, the mutant strain is modified with transcriptional units encoding a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant yeast strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids.

[0250] In an example to produce sialic acid and CMP-sialic acid, a yeast expression plasmid derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the TRP1 selection was modified as described in e.g., WQ22034067. To allow sialylated oligosaccharide production, the mutant S. cerevisiae strain producing CMP-sialic acid was further modified with one or more transcriptional unit(s) encoding one or more sialyltransferases. In a next step, the mutant strain is modified with transcriptional units encoding a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant yeast strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids.

[0251] Gene

[0252] Genes were expressed using synthetic constitutive promoters, as described by e.g., Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012), Redden and Alper (Nat. Commun. 2015, 6, 7810), Liu et al. (Microb. Cell Fact. 2020, 19, 38), Xu et al. (Microb. Cell Fact.2021, 20, 148) and Lee et al. (ACS Synth. Biol. 2015, 4(9), 975-986).

[0253] Cultivations conditions

[0254] In general, yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30°C. Starting from a single colony, a preculture was grown over night in 5 mL at 30°C, shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30°C with an orbital shaking of 200 rpm for 72h, or shorter of longer. At the end of the cultivation experiment samples were taken to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 60 min at 60°C before spinning down the cells (= whole broth concentration, i.e., intra- and extracellular sugar concentrations). C. Bacillus subtilis

[0255] Media

[0256] Two media are used to cultivate B. subtilis: i.e., a complex medium like a rich Luria Broth (LB) and a minimal medium for shake flask cultures. The LB medium consisted of 1% tryptone peptone (Difco), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR). Luria Broth agar (LBA) plates consisted of the LB media, with 12 g / L agar (Difco) added. The minimal medium contained 2.00 g / L (NH4)2SO4, 7.5 g / L KH2PO4, 17.5 g / L K2HPO4, 1.25 g / L Na-citrate, 0.25 g / L MgSO4.7H2O, 0.05 g / L tryptophan, from 10 up to 30 g / L glucose (or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose), 10 mL / L trace element mix and 10 mL / L Fe-citrate solution. The medium was set to a pH of 7 with 1 M KOH. Depending on the experiment, for the production of a saccharide, 0.30 g / L sialic acid, 0.30 g / L GIcNAc, 20 g / L lactose, 20 g / L 2'FL, 20 g / L 3-FL, 20 g / L Di FL, 20 g / L LN3, 20 g / L LNT, 20 g / L LNnT, 20 g / L LacNAc and / or 20 g / L LNB was additionally added to the medium. The trace element mix consisted of 0.735 g / L CaCI2.2H2O, 0.1 g / L MnCI2.2H2O, 0.033 g / L CuCI2.2H2O, 0.06 g / L CoCI2.6H2O, 0.17 g / L ZnCI2, 0.0311 g / L H3BO4, 0.4 g / L Na2EDTA.2H2O and 0.06 g / L Na2MoO4. The Fe-citrate solution contained 0.135 g / L FeCI3.6H2O, 1 g / L Na-citrate (Hoch 1973 PMC1212887). Complex medium, e.g., LB, was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic.

[0257] Strains, plasmids and mutations

[0258] B. subtilis 168 is used as available at the Bacillus Genetic Stock Center (Ohio, USA). Plasmids for gene deletion via Cre / lox are constructed as described by Yan et al. (Appl & Environm microbial, Sept 2008, p5556-5562). Gene disruption is done via homologous recombination with linear DNA and transformation via the electroporation as described by Xue et al. (J. microb. Meth. 34 (1999) 183-191). The method of gene knockouts is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). Integrative vectors as described by Popp et al. (Sci. Rep., 2017, 7, 15158) are used as expression vector and could be further used for genomic integrations if necessary. A suitable promoter for expression can be derived from the part repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

[0259] In an example, the mutant strain was derived from B. subtilis 168 and modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In a next step for LN3 production, the strain was modified to comprise a transcriptional unit for a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. LgtA from N. meningitidis (UniProt ID Q9JXQ6). In another example, the B. subtilis strain engineered for LN3 production is further modified with a transcriptional unit for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli 055:1-17 to produce LNT or with an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis to produce LNnT.

[0260] In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from B. subtilis 168 and modified as described e.g., in WO 2022 / 034067. To allow sialylated oligosaccharide production, the mutant B. subtilis strain producing CMP-sialic acid was further modified with one or more transcriptional unit(s) encoding one or more sialyltransferases. The strain is additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids.

[0261] In an example for fucosylated oligosaccharide production, the mutant strain was derived from B. subtilis 168 and modified as described e.g., in WQ22034069 to comprise one or more transcriptional unit(s) encoding one or more fucosyltransferases. Additionally, the mutant strain is further modified with a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids.

[0262] In an example to produce one or more fucosylated non-charged oligosaccharide(s), a B. subtilis strain is modified for production of GDP-fucose, LN3, LNT and / or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s).

[0263] In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, a B. subtilis strain is modified for production of CMP-sialic acid, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s).

[0264] In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, a B. subtilis strain is modified for production of CMP-sialic acid, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s). Cultivation conditions

[0265] A preculture was started from a cryovial or a single colony from an LBA plate, in 6 mL LB and was incubated overnight at 37 °C on an orbital shaker at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL minimal medium. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer. At the end of the cultivation experiment samples were taken to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 60 min at 60°C before spinning down the cells (= whole broth concentration, i.e., intra- and extracellular sugar concentrations).

[0266] D. Corynebacterium glutamicum

[0267] Media

[0268] Two different media are used, namely complex medium like e.g., a rich tryptone-yeast extract (TY) medium, and a minimal medium for shake flask (MMsf). The minimal medium uses a lOOOx stock trace element mix. Trace element mix consisted of 10 g / L CaCI2, 10 g / L FeSO4.7H2O, 10 g / L MnSO4.H2O, 1 g / L ZnSO4.7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCI2.6H2O, 0.2 g / L biotin (pH 7) and 0.03 g / L protocatechuic acid. The minimal medium for the shake flasks (MMsf) experiments contained 20 g / L (NH4)2SO4, 5 g / L urea, 1 g / L KH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4.7H2O, 42 g / L MOPS, from 10 up to 30 g / L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples and 1 ml / L trace element mix. Depending on the experiment, for the production of a saccharide, 0.30 g / L sialic acid, 0.30 g / L GIcNAc, 20 g / L lactose, 20 g / L 2'FL, 20 g / L 3-FL, 20 g / L DiFL, 20 g / L LN3, 20 g / L LNT, 20 g / L LNnT, 20 g / L LacNAc and / or 20 g / L LNB was additionally added to the medium. The TY medium consisted of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). TY agar (TYA) plates consisted of the TY media, with 12 g / L agar (Difco, Erembodegem, Belgium) added. Complex medium, e.g., TY, was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic.

[0269] Strains and mutations

[0270] Corynebacterium glutamicum was used as available at the American Type Culture Collection (ATCC 13032). Integrative plasmid vectors were made using the Cre / loxP technique as described by Suzuki et al. (Appl. Microbiol. BiotechnoL, 2005 Apr, 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et al. (Journal of Microbiological Methods 85, 2011, 155-163) are constructed for gene deletions, mutations and insertions. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (BiotechnoL Bioeng., 2013 Nov, 110(ll):2959-69). Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. In an example for LN3 production, a non-charged oligosaccharide, the mutant strain was derived from C. glutamicum and modified to comprise a transcriptional unit for a galactoside beta-1, 3-N- acetylglucosaminyltransferase like e.g. LgtA from N. meningitidis (UniProt ID Q9JXQ6). In a next step, the mutant strain is modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In another example the C. glutamicum strain engineered for LN3 production is further modified with a transcriptional unit for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli 055:1-17 to produce LNT or with an N-acetylglucosamine beta-1, 4- galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis to produce LNnT.

[0271] In an example for fucosylated oligosaccharide production, the mutant strain is derived from C. glutamicum and modified as described e.g., in WQ22034069 to comprise one or more transcriptional unit(s) encoding one or more fucosyltransferases. The strain is additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In an example to produce one or more fucosylated non-charged oligosaccharide(s), a C. glutamicum strain is modified for production of GDP-fucose, LN3, LNT and / or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s). In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from C. glutamicum and modified as described e.g., in WQ22034067. To allow sialylated oligosaccharide production, the mutant C. glutamicum strain producing CMP-sialic acid was further modified with one or more transcriptional unit(s) encoding one or more sialyltransferases. The strain is additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids.

[0272] In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, a C. glutamicum strain is modified for production of CMP-sialic acid, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s).

[0273] In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, a C. glutamicum strain is modified for production of CMP-sialic acid, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).

[0274] Cultivation conditions

[0275] A preculture was started from a cryovial or a single colony from a TY plate, in 6 mL TY and was incubated overnight at 37 °C on an orbital shaker at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL MMsf medium. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer. At the end of the cultivation experiment samples were taken to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 60 min at 60°C before spinning down the cells (= whole broth concentration, i.e., intra- and extracellular sugar concentrations).

[0276] E. Citrobacter werkmanii

[0277] Media

[0278] The cultivation medium used is as described by Maervoet et al. (Green Chem. 2012, 14, 2168-2179). Depending on the experiment, 0.30 g / L sialic acid, 0.30 g / L GIcNAc, 20 g / L lactose, 20 g / L 2'FL, 20 g / L 3- FL, 20 g / L DiFL, 20 g / L LN3, 20 g / L LNT, 20 g / L LNnT, 20 g / L LacNAc and / or 20 g / L LNB was additionally added to the medium.

[0279] Strains and mutations

[0280] Citrobacter werkmanii DSM17579 is available from the German Collection of Microorganisms and Cell Cultures Gmbh (DSMZ, Germany). All mutant strains were created as described by Maervoet et al. (Green Chem. 2012, 14, 2168-2179). In an example for LN3 production, the mutant strain was derived from C. werkmanii and modified with a genomic knock-out of the endogenous lactose permease LacY (UniProt ID A0A6I5AGA6) and modified to comprise a transcriptional unit for a galactoside beta-1, 3-N- acetylglucosaminyltransferase like e.g. LgtA from N. meningitidis (UniProt ID Q9JXQ6). In a next step, the mutant strain is modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In another example the C. werkmanii strain engineered for LN3 production is further modified with a transcriptional unit for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli O55:H7 to produce LNT or with an N-acetylglucosamine beta-1, 4- galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis to produce LNnT. In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from C. werkmanii and modified with a genomic knock-out of the endogenous lactose permease LacY (UniProt ID A0A6I5AGA6) and modified with constitutive transcriptional units comprising an L-glutamine— D-fructose-6-phosphate aminotransferase like e.g. glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)), a glucosamine 6-phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577), a phosphatase like e.g. SurE from E. coli (UniProt ID P0A840), an N- acylglucosamine 2-epimerase like e.g. AGE from B. ovatus (UniProt ID A7LVG6), an N-acetylneuraminate synthase like e.g. NeuB from N. meningitidis (UniProt ID E0NCD4) and an N-acylneuraminate cytidylyltransferase like e.g. NeuA from P. multocida (UniProt ID A0A849CI62). To allow sialylated oligosaccharide production, the mutant C. werkmanii strain producing CMP-sialic acid was further modified with one or more transcriptional unit(s) encoding one or more sialyltransferases. The strain is additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In an example for fucosylated oligosaccharide production, the mutant strain is derived from C. glutamicum with a genomic knock-out of the endogenous lactose permease LacY (UniProt ID A0A6I5AGA6) and modified with constitutive transcriptional units comprising a mannose-6-phosphate isomerase like e.g. manA from E. coli (UniProt ID P00946), a phosphomannomutase like e.g. manB from E. coli (UniProt ID P24175), a mannose-l-phosphate guanylyltransferase like e.g. manC from E. coli (UniProt ID P24174, sequence version 03, 19 July 2003), a GDP-mannose 4,6-dehydratase like e.g. gmd from E. coli (UniProt ID P0AC88) and a GDP-L-fucose synthase like e.g. fcl from E. coli (UniProt ID P32055 and one or more fucosyltransferases. The strain is additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the mutant strain is modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In an example to produce one or more fucosylated non-charged oligosaccharide(s), a C. werkmanii strain is modified for production of GDP-fucose, LN3, LNT and / or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, a C. werkmanii strain is modified for production of CMP-sialic acid, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, a C. glutamicum strain is modified for production of CMP-sialic acid, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).

[0281] Cultivation conditions

[0282] A preculture was started from a cryovial or a single colony from an LBA plate, in 6 mL LB and was incubated overnight at 37 °C on an orbital shaker at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL minimal medium. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer. At the end of the cultivation experiment samples were taken to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 60 min at 60°C before spinning down the cells (= whole broth concentration, i.e., intra- and extracellular sugar concentrations).

[0283] F. Chlamydomonas reinhardtii

[0284] Media and cultivation

[0285] Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7). The TAP medium uses a lOOOx stock Hutner's trace element mix. Hutner's trace element mix consisted of 50 g / L Na2EDTA.H2O (Titriplex III), 22 g / L ZnSO4.7H2O, 11.4 g / L H3BO3, 5 g / L MnCI2.4H2O, 5 g / L FeSO4.7H2O, 1.6 g / L CoCI2.6H2O, 1.6 g / L CuSO4.5H2O and 1.1 g / L (NH4)6MoO3. The TAP medium contained 2.42 g / L Tris (tris(hydroxymethyl)aminomethane), 25 mg / L salt stock solution, 0.108 g / L K2HPO4, 0.054 g / L KH2PO4 and 1.0 mL / L glacial acetic acid. The salt stock solution consisted of 15 g / L NH4CL, 4 g / L MgSO4.7H2O and 2 g / L CaCI2.2H2O. Depending on the experiment, 0.30 g / L sialic acid, 0.30 g / L GIcNAc,

[0286] 20 g / L lactose, 20 g / L 2'FL, 20 g / L 3-FL, 20 g / L DiFL, 20 g / L LN3, 20 g / L LNT, 20 g / L LNnT, 20 g / L LacNAc and / or 20 g / L LNB was additionally added to the medium. Medium was sterilized by autoclaving (121°C,

[0287] 21 min). For stock cultures on agar slants TAP medium was used containing 1% agar (of purified high strength, 1000 g / cm2).

[0288] Cells of C. reinhardtii were cultured in selective TAP-agar plates at 23 + / - 0.5°C under 14 / 10 h I ight / dark cycles with a light intensity of 8000 Lx. Cells were analysed after 5 to 7 days of cultivation. For high-density cultures, cells could be cultivated in closed systems like e.g., vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat panel photobioreactors as described by Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827). Strains, plasmids and mutations

[0289] C. reinhardtii wild-type strains 21gr (CC-1690, wild-type, mt+), 6145C (CC-1691, wild-type, mt-), CC-125 (137c, wild-type, mt+), CC-124 (137c, wild-type, mt-) as available from the Chlamydomonas Resource Center (https: / / www.chlamycollection.org) (University of Minnesota, U.S.A) were used. Expression plasmids originated from pSH03, as available from the Chlamydomonas Resource Center. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived from e.g., Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modification (like gene knock-out or gene replacement) can be carried using the Crispr-Cas technology as described e.g., by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469). Transformation via electroporation was performed as described by Wang et al. (Biosci. Rep. 2019, 39: BSR2018210) and as described like e.g., in WO22034067 or in WO22034069.

[0290] In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067. In an example for production of sialylated oligosaccharides, C. reinhardtii cells are modified with a CMP-sialic acid transporter like e.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localised sialyltransferase selected from species like e.g., Homo sapiens, Mus musculus, Rattus norvegicus. The cells are additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the cells are modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In an example for GDP-fucose synthesis, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WQ22034067. In an example for fucosylation, C. reinhardtii cells can be modified with an expression plasmid comprising a constitutive transcriptional unit for an alpha-1, 2-fucosyltransferase and / or an alpha-1, 3-fucosyltransferase. The cells are additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the cells are modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re- introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In an example for UDP-galactose synthesis, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WQ22034067. In an example for LN3 production, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WQ22034067 to comprise a transcriptional unit for a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (UniProt ID Q9JXQ6). In an example for LNT or LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli O55:H7 or an N-acetylglucosamine beta-1, 4- galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis, respectively. The cells are additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the cells are modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids. In an example to produce one or more fucosylated non-charged oligosaccharide(s), a C. reinhardtii strain is modified for production of GDP-fucose, UDP-galactose, LN3, LNT and / or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s) like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 10) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having betagalactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6- sialyltransferase (PdST6) from P. damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 11) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6- sialyltransferase activity. In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).

[0291] G. Animal cells

[0292] Isolation of mesenchymal stem cells from adipose tissue of different animals

[0293] Fresh adipose tissue is obtained from slaughterhouses (e.g., cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs) or liposuction (e.g., in case of humans, after informed consent) and kept in phosphate buffer saline supplemented with antibiotics. Enzymatic digestion of the adipose tissue is performed followed by centrifugation to isolate mesenchymal stem cells. The isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions, e.g., 37°C, 5% CO2. The initial culture medium includes DMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% foetal bovine serum), and 1% antibiotics. The culture medium is subsequently replaced with 10% FBS (foetal bovine serum)-supplemented media after the first passage. For example, Ahmad and Shakoori (2013, Stem Cell Regen. Med. 9(2): 29-36), which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example. Isolation of mesenchymal stem cells from milk

[0294] This example illustrates isolation of mesenchymal stem cells from milk collected under aseptic conditions from human or any other mammal(s) such as described herein. An equal volume of phosphate buffer saline is added to diluted milk, followed by centrifugation for 20 min. The cell pellet is washed thrice with phosphate buffer saline and cells are seeded in cell culture flasks in DMEM-F12, RPMI, and Alpha-MEM medium supplemented with 10% foetal bovine serum and 1% antibiotics under standard culture conditions. For example, Hassiotou et al. (2012, Stem Cells. 30(10): 2164-2174), which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example.

[0295] Differentiation of stem cells using 2D and 3D culture systems

[0296] The mesenchymal cells isolated from adipose tissue of different animals or from milk as described above can be differentiated into mammary-like epithelial and luminal cells in 2D and 3D culture systems. See, for example, Huynh et al. 1991. Exp Cell Res. 197(2): 191 -199; Gibson et al. 1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchford et al. 1999; Animal Cell Technology': Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5): C348 - C356; each of which is incorporated herein by reference in their entireties for all purposes.

[0297] For 2D culture, the isolated cells were initially seeded in culture plates in growth media supplemented with 10 ng / mL epithelial growth factor and 5 pg / mL insulin. At confluence, cells were fed with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U / mL penicillin, 100 ug / mL streptomycin), and 5 pg / mL insulin for 48h. To induce differentiation, the cells were fed with complete growth medium containing 5 pg / mL insulin, 1 pg / mL hydrocortisone, 0.65 ng / mL triiodothyronine, 100 nM dexamethasone, and 1 pg / mL prolactin. After 24h, serum is removed from the complete induction medium.

[0298] For 3D culture, the isolated cells were trypsinized and cultured in Matrigel, hyaluronic acid, or ultra- low attachment surface culture plates for six days and induced to differentiate and lactate by adding growth media supplemented with 10 ng / mL epithelial growth factor and 5 pg / mL insulin. At confluence, cells were fed with growth medium supplemented with 2% foetal bovine serum, 1% penicillin-streptomycin (100 U / mL penicillin, 100 ug / mL streptomycin), and 5 pg / mL insulin for 48h. To induce differentiation, the cells were fed with complete growth medium containing 5 pg / mL insulin, 1 pg / mL hydrocortisone, 0.65 ng / mL triiodothyronine, 100 nM dexamethasone, and 1 pg / mL prolactin. After 24h, serum is removed from the complete induction medium. Method of making mammary-like cells

[0299] In a next step, the cells are brought to induced pluripotency by reprogramming with viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc. The resultant reprogrammed cells are then cultured in Mammocult media (available from Stem Cell Technologies), or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make them mammary-like, from which expression of select milk components can be induced. Alternatively, epigenetic remodelling is performed using remodelling systems such as CRISPR / Cas9, to activate select genes of interest, such as casein, a- lactalbumin to be constitutively on, to allow for the expression of their respective proteins, and / or to down-regulate and / or knock-out select endogenous genes as described e.g., in WO21067641, which is incorporated herein by reference in its entirety for all purposes. In an example for production of one or more oligosaccharide(s), isolated mesenchymal cells re-programmed into mammary-like cells are modified via CRISPR-CAS as described e.g., in WO22034067, W022034070 and WO22034075. The cells are additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. Alternatively, and / or additionally, the cells are modified for overexpression of a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05 wherein said overexpression is obtained by well-known methods such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re- introduction of an expression module at euchromatin, usage of high-copy-number plasmids.

[0300] Cultivation

[0301] Completed growth media includes high glucose DMEM / F12, 10% FBS, 1% NEAA, 1% pen / strep, 1% ITS-X, 1% F-Glu, 10 ng / mL EGF, and 5 pg / mL hydrocortisone. Completed lactation media includes high glucose DMEM / F12, 1% NEAA, 1% pen / strep, 1% ITS-X, 1% F-Glu, 10 ng / mL EGF, 5 pg / mL hydrocortisone, and 1 pg / mL prolactin (5ug / mL in Hyunh 1991). Cells are seeded at a density of 20,000 cells / cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media. Upon exposure to the lactation media, the cells start to differentiate and stop growing. Within about a week, the cells start secreting lactation product(s) such as milk lipids, lactose, casein and whey into the media. A desired concentration of the lactation media can be achieved by concentration or dilution by ultrafiltration. A desired salt balance of the lactation media can be achieved by dialysis, for example, to remove unwanted metabolic products from the media. Hormones and other growth factors used can be selectively extracted by resin purification, for example the use of nickel resins to remove His-tagged growth factors, to further reduce the levels of contaminants in the lactated product.

[0302] H. Optical density

[0303] Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10M microplate reader, Tecan, Switzerland). The maximum growth speed (mumax) was calculated based on the observed optical densities at 600nm using the R package grofit. / . Heterologous and homologous expression

[0304] Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: IDT, Twist Bioscience, DNA2.0 or Gen9. Proteins described in present disclosure are summarized in Table 1. Unless stated otherwise, the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database version release 2021_03 of 09 June 2021. Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

[0305] Table 1.

[0306] Overview of proteins with corresponding SEQ ID NOs or UniProt IDs (sequence version 01, UniProt Database 2021_03 of 09 June 2021) as described in present invention

[0307] (a)Sequence version 02 (01 Oct 1993) as present in the UniProt Database 2021_03 of 09 June 2021

[0308] (b)Sequence version 03 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021

[0309] (c)Sequence version 04 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021 (d)Sequence version 02 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021

[0310] (e)Sequence version 02 (01 Dec 2000) as present in the UniProt Database 2021_03 of 09 June 2021

[0311] (f) Sequence version 03 (19 July 2003) as present in the UniProt Database 2021_03 of 09 June 2021 J. Analytical analysis

[0312] Standards such as but not limited to sucrose, lactose, 3'SL and 6'SLwere purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards.

[0313] Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (Rl) detection. A volume of 0.7 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm) column with an Acquity UPLC BEH Amide VanGuard column, 130 A, 2. lx 5 mm. The column temperature was 50 °C. The mobile phase consisted of a % water and % acetonitrile solution to which 0.2 % triethylamine was added. The method was isocratic with a flow of 0.130 mL / min. The ELS detector had a drift tube temperature of 50 °C and the N2 gas pressure was 50 psi, the gain 200 and the data rate 10 pps. The temperature of the Rl detector was set at 35 °C. Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection. A volume of 0. 5 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm). The column temperature was 50 °C. The mobile phase consisted of a mixture of 70 % acetonitrile, 26 % ammonium acetate buffer (150 mM) and 4 % methanol to which 0.05 % pyrrolidine was added. The method was isocratic with a flow of 0.150 mL / min. The temperature of the Rl detector was set at 35 °C. Both neutral and sialylated sugars were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection. A volume of 0.5 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm). The column temperature was 50°C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow of 0.260 mL / min. The temperature of the Rl detector was set at 35°C. For analysis on a UPLC with a fluorescent detector, sugars were labelled with 2-aminobenzamide (2AB) and a 2-Methylpyridine borane complex. The Acquity UPLC system (Waters) was used with two Acquity UPLC BEH Amide columns (2. lx 50mm; 1.7pm, Waters), one serving as Guard, and one used for separation with a gradient of Acetonitrile (A) and 100 mM ammonium formate and 0.05% formic acid (B) wherein the gradient used was 1) from 0 to 0.25 min 75% A, 25% B, 2) from 0.25 to 2.15 min changing A from 75% to 60% and B from 25% to 40%, 3) from 2.15 min to 2.25 min maintaining the percentages of A and B, 4) from 2.25 to 2.5 min changing A from 60 to 75%, B from 40 to 25%, and 5) from 2.5 to 5 min maintaining the percentages of A and B. For analysis on a mass spectrometer, a Waters Xevo TQ-MS with Electron Spray Ionisation (ESI) was used with a desolvation temperature of 450 °C, a nitrogen desolvation gas flow of 650 L / h and a cone voltage of 20 V. The MS was operated in selected ion monitoring (SIM) in negative mode for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1 x 100 mm; 3 pm) on 35 °C. A gradient was used wherein eluent A was ultrapure water with 0.1 % formic acid and wherein eluent B was acetonitrile with 0.1 % formic acid. The oligosaccharides were separated in 55 min using the following gradient: an initial increase from 2 to 12 % of eluent B over 21 min, a second increase from 12 to 40 % of eluent B over 11 min and a third increase from 40 to 100 % of eluent B over 5 min. As a washing step 100 % of eluent B was used for 5 min. For column equilibration, the initial condition of 2 % of eluent B was restored in 1 min and maintained for 12 min.

[0314] Example 4. Production of LNT or LNnT with a modified E. coli host

[0315] An E. coli K12 MG1655 strain was modified as described in Example 3 comprising genomic knock-outs of the E. coli genes LacY, LacZ, nagB, galT and ushA and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6) and the galactoside beta-1, 3-N-acetylglucosaminyltransferase LgtA (UniProt ID Q9JXQ6) from N. meningitidis. In order to produce LNT or LNnT, the mutant strain was further modified with a genomic knock-in of a constitutive transcriptional unit containing either the N-acetylglucosamine beta-1, 3-galactosyltransferase wbgO (Uniprot ID D3QY14) from E. coli 055:1-17 or the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis, respectively. The novel strains were evaluated in a growth experiment for production of LN3 and either LNT or LNnT according to the culture conditions provided in Example 4, in which the strains were cultivated in minimal medium supplemented with 15 g / L sucrose and 20 g / L Lactose. Reference strains were used with the same genetic make-up as the novel mutant strains, either adapted for LNT or LNnT synthesis, and no LacY KO, i.e. still having an active E. coli LacY gene. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 3. For each strain the measured LN3 and LNT or LNnT concentration was averaged over all biological replicates. Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI was determined by dividing the LN3, LNT or LNnT concentrations, measured in the whole broth, by the biomass. The biomass is empirically determined to be approximately l / 3rd of the optical density measured at 600 nm. The data is summarized in Table 2. The experiment demonstrated that the mutant strains, having no LacY gene, were able to produce LN3 and either LNT or LNnT in comparable amounts as their respective reference strains.

[0316] Table 2.

[0317] CPI data obtained in a growth experiment with a mutant E. coli LacY KO strain adapted for LNT or LNnT production (mutant strain) or a reference strain adapted for LNT or LNnT production and expressing an active LacY gene. The CPI data refer to LN3, LNT or LNnT production in whole broth samples. The growth experiment was performed according to the culture conditions provided in Example 4, in which the cultivation medium was supplemented with 15 g / L sucrose and 20 g / L lactose.

[0318] Example 5. Evaluation of LNT production with a modified E. coli host

[0319] The mutant E. coli K12 MG1655 strain adapted for LNT production and having a LacY KO as described in Example 5 producing LNT was further modified with a knockout of the E. coli melB gene. The novel strain was evaluated in a growth experiment for production of LN3 and LNT according to the culture conditions provided in Example 3, in which the strains were cultivated in minimal medium supplemented with 15 g / L sucrose and 20 g / L Lactose. A reference strain was used with the same genetic make-up as the novel mutant strain but still having an active copy of the E. coli melB gene. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 3. For each strain the measured LN3 and LNT concentration was averaged over all biological replicates. Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI was determined by dividing the LN3 and LNT concentrations, measured in the whole broth, by the biomass. The data is summarized in Table 3. The experiment demonstrated that the mutant strain, having knock-outs for E. coli melB and LacY, produced no LN3 and very low levels of LNT compared to its reference strain having only the LacY KO but still expressing E. coli melB pointing towards the importance of having an active melB in the production host for production of LN3 and / or LNT.

[0320] Table 3.

[0321] CPI data obtained in a growth experiment with a mutant E. coli LacY KO and melB KO strain adapted for LNT production (Mutant strain) or a reference strain adapted for LNT production and having a LacY KO but still expressing E. coli melB. The CPI data refer to LN3 and LNT production in whole broth samples. The growth experiment was performed according to the culture conditions provided in Example 3, in which the cultivation medium was supplemented with 15 g / L sucrose and 20 g / L lactose.

[0322] Example 6. Evaluation of LNT production with a modified E. coli host

[0323] An E. coli K12 MG1655 strain is modified as described in Example 3 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and LacY and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter CscB from E. coli W (UniProt ID E0IXR1), the fructose kinase Frk from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase LgtA (UniProt ID Q9JXQ6) from N. meningitidis and the N-acetylglucosamine beta-1, 3-galactosyltransferase wbgO (Uniprot ID D3QY14) from E. coli O55:H7. In a next step, the mutant strain is transformed with an expression plasmid containing a constitutive transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. The novel strains are evaluated in a growth experiment for production of LN3 and LNT according to the culture conditions provided in Example 3, in which the strains are cultivated in minimal medium supplemented with 15 g / L sucrose and 20 g / L lactose. Regular samples are taken and evaluated for production of LN3 and LNT.

[0324] Example 7. Evaluation of LNnT production with a modified E. coli host

[0325] An E. coli K12 MG1655 strain is modified as described in Example 3 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT, ushA and LacY and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter CscB from E. coli W (UniProt ID E0IXR1), the fructose kinase Frk from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase LgtA (UniProt ID Q9JXQ6) from N. meningitidis and the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis. In a next step, the mutant strain is transformed with an expression plasmid containing a constitutive transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. The novel strains are evaluated in a growth experiment for production of LN3 and LNnT according to the culture conditions provided in Example 3, in which the strains are cultivated in minimal medium supplemented with 15 g / L sucrose and 20 g / L lactose. Regular samples are taken and evaluated for production of LN3 and LNnT.

[0326] Example 8. Evaluation of production of 3-FL or DiFL with a modified E. coli host

[0327] An E. coli K12 MG1655 strain was modified for production of GDP-fucose as described in Example 3 comprising a genomic knock-out of the E. coli gene LacZ and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6). In a next step, the mutant strain was further modified with a knockout for the E. coli LacY gene. This novel strain was further transformed with an expression plasmid containing a constitutive transcriptional unit for the alpha-1, 3-fucosyltransferase from B. psittacipulmonis (UniProt ID A0A077DGH3). The novel strain was evaluated in a growth experiment for production of 3-FL or DiFL according to the culture conditions provided in Example 3, in which the strains were cultivated in minimal medium supplemented with (a) 15 g / L sucrose and 20 g / L Lactose or (b) 15 g / L sucrose and 20 g / L 2'FL, respectively. A reference strain was used with the same genetic make-up as the novel mutant strain but without a LacY KO, i.e. still having an active E. coli LacY gene. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 3. For each strain the measured 3FL and DiFL concentration was averaged over all biological replicates. Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI was determined by dividing the 3FL and DiFL concentrations, measured in the whole broth by the biomass. The data is summarized in Table 4. The experiment demonstrated that the mutant strain was (1) not able to produce 3-FL when cultivated in medium supplemented with lactose or (2) not able to produce DiFL when cultivated in medium supplemented with 2'FL, whereas the reference strain was (1) able to produce 3-FL when cultivated in medium supplemented with lactose and (2) able to produce DiFL when cultivated in medium supplemented with 2'FL.

[0328] Table 4.

[0329] CPI data obtained in a growth experiment with a mutant E. coli LacY KO strain adapted for 3-FL or DiFL production (mutant strain) or a reference strain adapted for 3-FL or DiFL production and expressing an active LacY gene. The CPI data refer to 3-FL and DiFL production in whole broth samples. The growth experiment was performed according to the culture conditions provided in Example 1, in which the cultivation medium was supplemented (1) with 15 g / L sucrose and 20 g / L lactose (= condition 1) or (2) with 15 g / L sucrose and 20 g / L 2'FL (= condition 2).

[0330] Example 9. Evaluation of DiFL production with a modified E. coli host

[0331] A mutant E. coli K12 MG1655 strain modified for production of LNT as described in Example 3 was further modified by genomic knock-in of constitutive transcriptional units containing the phosphomannomutase manB from E. coli (UniProt ID P24175), the mannose-l-phosphate guanylyltransferase manC from E. coli (UniProt ID P24174), the GDP-mannose 4,6-dehydratase gmd from E. coli (UniProt ID P0AC88) and the GDP-L-fucose synthase fcl from E. coli (UniProt ID P32055). In a next step, the mutant strain was further modified with a genomic knock-out for (1) the E. coli LacY gene or (2) both the E. coli LacY and melB gene. The novel strains were finally transformed with an expression plasmid containing a constitutive transcriptional unit for the alpha-1, 3-fucosyltransferase from P. catoniae (UniProt ID Z4WWI2). The novel strains were evaluated in a growth experiment for production of DiFL according to the culture conditions provided in Example 3, in which the strains were cultivated in minimal medium supplemented with 15 g / L sucrose and 20 g / L 2'FL. A reference strain was used with the same genetic make-up as the novel mutant strains but without a LacY and melB KO, i.e. still having an active E. coli LacY and melB gene. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed as described in Example 4. For each strain the measured DiFL concentration was averaged over all biological replicates. Also, a dilution of the cultures was made to measure the optical density at 600 nm. The cell performance index or CPI was determined by dividing the DiFL concentrations, measured in the whole broth by the biomass. The data is summarized in Table 5. The experiment demonstrated that both mutant strains were not able to produce DiFL when cultivated in medium supplemented with 2'FL, in contrast to the reference strain that did produce DiFL in said conditions. Herewith, it was noticed that the DiFL production dropped in mutant strain 1 having a LacY KO but still expressing an active E. coli melB and that the further knock-out of the me / B gene in the mutant strain 1 did not influence DiFL production, demonstrating that E. coli melB is not involved in the uptake of 2'FL inside the cell and that 2'FL is imported by action of the E. coli LacY in the strain.

[0332] Table 5.

[0333] CPI data obtained in a growth experiment with mutant E. coli strains adapted for production of DiFL and with (1) a LacY KO (Mutant strain 1) or (2) a LacY KO and melB KO (Mutant strain 2) or a reference strain adapted for DiFL production and expressing an active E. coli LacY and melB gene. The CPI data refer to DiFL production in whole broth samples. The growth experiment was performed according to the culture conditions provided in Example 1, in which the cultivation medium was supplemented with 15 g / L sucrose and 20 g / L 2'FL.

[0334] Example 10. Evaluation of 2'FL production with a modified E. coli host

[0335] An E. coli K12 MG1655 strain is modified for GDP-fucose production as described in Example 3 comprising genomic knock-outs of the E. coli genes lacZ and LacY and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter CscB from E. coli W (UniProt ID E0IXR1), the fructose kinase Frk from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6). In a next step, the mutant strain is modified with a genomic knock-in of a constitutive transcriptional unit for a membrane protein selected from the list consisting of SEQ. ID NO 01, 02, 03, 04 and 05. In a final step, the mutant strains are transformed with an expression plasmid containing a constitutive transcriptional unit for the alpha-1, 2-fucosyltransferase (HpFutC) from H. pylori (UniProt ID Q9X435). The novel strains are evaluated in a growth experiment for production of 2'FL according to the culture conditions provided in Example 3, in which the strains are cultivated in minimal medium supplemented with 15 g / L sucrose and 20 g / L lactose. Regular samples are taken and evaluated for production of 2'FL. Example 11. Evaluation of 3-SL production with a modified E. coli host

[0336] An E. coli K12 MG1655 strain is modified for CMP-sialic acid production as described in Example 3 comprising genomic knock-outs of the E. coli genes LacZ and LacY and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter CscB from E. coli W (UniProt ID E0IXR1), the fructose kinase Frk from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase BaSP from B. adolescentis (UniProt ID A0ZZH6). In a next step, the mutant strain is modified with a genomic knock-in of a constitutive transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. In a final step, the mutant strains are transformed with an expression plasmid containing a constitutive transcriptional unit for the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3). The novel strains are evaluated in a growth experiment for production of 3-SL according to the culture conditions provided in Example 3, in which the strains are cultivated in minimal medium supplemented with 15 g / L sucrose and 20 g / L lactose. Regular samples are taken and evaluated for production of 3-SL.

[0337] Example 12. Evaluation of production of human milk oligosaccharide(s) with modified E. coli hosts when evaluated in a fed-batch fermentation process with sucrose and lactose

[0338] The mutant E. coli strains as described in Examples 4 to 11 are selected for further evaluation in fed-batch fermentation processes. Fed-batch fermentations at bioreactor scale are performed as described in Example 3. Sucrose is used as a carbon source and lactose is added in the batch medium. During fed- batch, sucrose is added via an additional feed. In contrast to the cultivation experiments that are described herein and wherein only end samples were taken at the end of cultivation (i.e., 72 hours as described herein), regular broth samples are taken at several time points during the fermentation process and the production of the desired oligosaccharide(s) is measured using UPLC as described in Example 3.

[0339] Example 13. Evaluation of production of 6'SL with a modified S. cerevisiae host

[0340] A S. cerevisiae strain, lacking a lactose permease, is modified for production of CMP-sialic acid and for expression of a sialyltransferase with a yeast expression plasmid containing the TRP1 selection marker and constitutive transcriptional units for the L-glutamine— D-fructose-6-phosphate aminotransferase glmS from E. coli (UniProt ID P17169, sequence version 04, 23 Jan 2007), the phosphatase SurE from E. coli (UniProt ID P0A840), the N-acylglucosamine 2-epimerase AGE from B. ovatus (UniProt ID A7LVG6), the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4), the N- acylneuraminate cytidylyltransferase NeuA from P. multocida (UniProt A0A849CI62) and the alpha-2, 6- sialyltransferase PdST6 from P. damselae (UniProt ID 066375). In a next step, the strain is modified with a constitutive transcriptional unit for a membrane protein selected from the list comprising SEQ ID NO 01, 02, 03, 04 and 05. The novel strains are evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 3, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors. The strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of sialic acid and 6'SL is analysed on UPLC.

[0341] Example 14. Evaluation of production of sialic acid and 3'SL with a modified B. subtilis host

[0342] In a first step, a B. subtilis strain, lacking a lactose permease, is modified with genetic knockouts of the genes nagA, nagB and gamA and with genetic knock-ins of constitutive transcriptional units for the native fructose-6-P-aminotransferase glmS (UniProt ID P0CI73), the UDP-N-acetylglucosamine 2-epimerase neuC from C. jejuni (UniProt ID Q93MP8), and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4). In a next step, the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferasePmultST3 from P. multocida (UniProt ID Q9CLP3). In a next step, the strain is modified with a constitutive transcriptional unit for a membrane protein selected from the list comprising SEQ ID NO 01, 02, 03, 04 and 05. The novel strains are evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 3, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors. The strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of sialic acid and 3'SL is analysed on UPLC.

[0343] Example 15. Evaluation of production of sialic acid and 3'SL with a modified C. glutamicum host

[0344] A mutant C. glutamicum strain, lacking a lactose permease, is created by genetic knockouts of the genes nagA, nagB and gamA and by overexpressing the native fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 03, 23 Jan 2007), the UDP-N-acetylglucosamine 2-epimerase neuC from C. jejuni (UniProt ID Q93MP8), and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4). In a next step, the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3). In a next step, the strain is modified with a constitutive transcriptional unit for a membrane protein selected from the list comprising SEQ ID NO 01, 02, 03, 04 and 05. The novel strains are evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 3, using MMsf medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of sialic acid and 3'SL.

[0345] Example 16. Evaluation of production of 3-FL with a modified C. werkmanii host

[0346] A mutant C. werkmanii strain is modified as described in Example 3 with a genomic knock-out of the endogenous lactose permease LacY (UniProt ID A0A6I5AGA6) and further modified with constitutive transcriptional units for the E. coli genes mannose-6-phosphate isomerase manA (UniProt ID P00946), the phosphomannomutase manB (UniProt ID P24175), the mannose-l-phosphate guanylyltransferase manC (UniProt ID P24174, sequence version 03, 19 July 2003), the GDP-mannose 4,6-dehydratase gmd (UniProt ID P0AC88), the GDP-L-fucose synthase fcl (UniProt ID P32055 and for the al,3-fucosyltransferase from B. psittacipulmonis (UniProt ID A0A077DGH3). The strain is additionally modified to comprise a transcriptional unit for a membrane protein selected from the list consisting of SEQ ID NO 01, 02, 03, 04 and 05. The novel strains are evaluated in a growth experiment for production of 3-FL according to the culture conditions provided in Example 3, using culture medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of 3-FL.

[0347] Example 17. Evaluation of LNT production with a modified E. coli host

[0348] An E. coli K12 MG1655 strain was modified as described in Example 3 comprising genomic knock-outs of the E. coli genes lacZ, nagB, galT and ushA, melB and LacY and genomic knock-ins of constitutive transcriptional units containing the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6), the galactoside beta-1, 3-N-acetylglucosaminyltransferase LgtA (UniProt ID Q9JXQ6) from N. meningitidis and the N-acetylglucosamine beta-1, 3-galactosyltransferase wbgO (Uniprot ID D3QY14) from E. coli 055:1-17.

[0349] In a next step, the mutant strain was transformed with an expression plasmid containing a constitutive transcriptional unit for melB (SEQ ID NO 02) from E. coli. The novel strain was evaluated in a growth experiment for production of LN3 and LNT according to the culture conditions provided in ...

Claims

Claims1. Method for importing a disaccharide inside a cell, said method comprising: providing a cell, metabolically engineering said cell by i) introducing and expressing, and / or ii) overexpressing a membrane protein, cultivating and / or incubating said cell, preferably in a cultivation medium comprising said disaccharide, preferably a single cell, under conditions permissive to express said membrane protein and to import said disaccharide inside the cell, characterized in that i) said disaccharide is selected from the list consisting of or consisting essentially of lactose, lactulose, N-acetyllactosamine and lacto-N-biose, and ii) said membrane protein a) has disaccharide importing activity across the membrane of said cell, b) originates from the major facilitator superfamily (MFS) of transporters, and c) comprises a polypeptide sequence comprising: o the conserved domain CD-INB1 with sequence [AGSV][HKNQR][CDEGNQTV] wherein the second amino acid residue is aligned to Lys 18 in SEQ ID NO 01, and o the conserved domain CD-INB2 with sequence [DEQ]XXX[FWY] wherein the first amino acid residue is aligned to Aspl24 in SEQ ID NO 01 and wherein X can be any amino acid residue.

2. Method according to claim 1, wherein said membrane protein:(a) comprises a polypeptide sequence comprising: an IPR domain selected from the list consisting of or consisting essentially of IPR001927, IPR018043, IPR039672 and IPR036259 domain as defined by InterPro 98.0 as released on 25th January 2024, the conserved domain cdl7332 domain as defined by the Conserved Domain Database CDD3.20 as released on 6th October 2022, the PANTHER domain PTHR11328 as defined by PANTHER 18.0 as released on 17th September 2023, and / or the PFAM domain PF13347 as defined by PFAM 32.0 as released in Sept 2018, and(b) is any one of SEQ ID NOs 02, 01, 03, 04 or 05, a functional homolog or functional fragment of any one of said SEQ ID NOs 02, 01, 03, 04 or05 having disaccharide importing activity, ora sequence having at least 40% sequence identity, preferably at least 80% sequence identity, to any one of the full-length amino acid sequences as represented by any one of SEQ. ID NOs 02, 01, 03, 04 or 05, and having disaccharide importing activity.

3. Method according to any one of previous claims, wherein said membrane protein is any one of an endogenous protein, a homologous protein or a heterologous protein of said cell.

4. Method according to any one of the previous claims, wherein said cell further comprises a pathway for production of a saccharide, an oligosaccharide, a milk oligosaccharide, or a human milk oligosaccharide, said method comprising a step of cultivating and / or incubating said cell under conditions permissive to express said membrane protein and to produce said saccharide.

5. Method according to claim 4, wherein said cell: comprises and / or is genetically engineered to comprise a pathway selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway, comprises at least one pathway selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway wherein at least one of said pathway(s) has / have been genetically engineered, possesses, expresses and / or overexpresses one or more glycosyltransferase(s) selected from the list consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N- acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N- acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl- beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases, is capable to produce and / or produces one or more nucleotide-activated sugars, is genetically engineered for production of one or more of nucleotide-activated sugar(s), comprises a pathway for the synthesis of a nucleotide-activated sugar selected from the list consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N- acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2- acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc orUDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N- acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP- Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), CMP-2-keto- 3-deoxymanno-octulonic acid (CMP-KDO), GDP-rhamnose and UDP-xylose, possesses, expresses and / or overexpresses one or more genes selected from the list consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L- glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N- acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GIcNAc 2- epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6- phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N- acetylglucosamine-l-phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase, Neu5Ac synthase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9- phosphate synthase, N-acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, d-arabinose 5-phosphate isomerase, KDO-8P synthase, KDO 8- phosphate phosphatase, CMP-KDO synthetase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1- phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N- acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, and / or comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and / or required for the synthesis of said saccharide.

6. Method according to any one of claims 4 or 5, wherein said saccharide is selected from the list consisting of or consisting essentially of monosaccharide; phosphorylated monosaccharide; activated monosaccharide; disaccharide; oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); sialylated milk oligosaccharide; neutral (non-charged) milk oligosaccharide; fucosylated milk oligosaccharide; non-fucosylated neutral (noncharged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylatedneutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non- fucosylated neutral (non-charged) human milk oligosaccharide; O-antigen; enterobacterial common antigen (ECA); the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; an antigen of the human ABO blood group system; an animal oligosaccharide; animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide; plant oligosaccharide selected from the list consisting of N-glycans and O-glycans; fucosylated oligosaccharide; fucosylated oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N- fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N- neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose; sialylated oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N- hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N- neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N- neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N-acetylglucosamine containing neutral (non-charged) saccharide; N-acetylglucosamine containing neutral (non-charged) saccharide selected from the list comprising lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N- hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N- hexaose, difucosyl-lacto-N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH II), difucosyl-para-lacto- N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N-trifucoheptaose; N- acetylglucosamine containing saccharide; N-acetyllactosamine containing saccharide; lacto-N-biose containing saccharide; non-fucosylated neutral (non-charged) saccharide; chitosan; chitosan comprising oligosaccharide; heparosan; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan sulphate; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.

7. Method according to any one of claims 4 to 6, wherein said cell: is capable to produce and / or produces said saccharide from one or more precursor(s), is capable to produce and / or produces said saccharide from lactose, is capable to produce and / or produces at least one precursor that is used to produce said saccharide, is capable to produce and / or produces all precursors that are used to produce said saccharide,is genetically engineered for the production of at least one precursor that is used to produce said saccharide, and / or is genetically engineered for the production of all precursors that are used to produce said saccharide.

8. Method according to claim 7 , wherein at least one of said one or more precursor(s) is internalized in said cell via (1) one or more transporter protein(s) and / or (2) said membrane protein.

9. Method according to any one of previous claims, wherein said cell is: a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell, an E. coli or yeast with a lactose permease positive phenotype, an E. coli or yeast with a lactose permease positive phenotype wherein said lactose permease is coded by the gene LacY or LAC12, respectively, an E. coli or yeast with a lactose permease negative phenotype, an E. coli or yeast with a lactose permease negative phenotype wherein said lactose permease is coded by the gene LacY or LAC12, respectively, an E. coli or yeast with a knocked-out or knocked-down lactose permease, and / or an E. coli or yeast with a knocked-out or knocked-down lactose permease wherein said lactose permease is coded by the gene LacY or LAC12, respectively.

10. Method according to any one of claims 4 to 9, wherein the cultivation or incubation medium used in said cultivation or incubation, respectively, comprises one or more precursor(s) that is / are used for production of said saccharide.

11. Method according to any one of claims 4 to 10, wherein said cell produces 25 g / L or more of said saccharide in the whole broth and / or supernatant and / or wherein said saccharide in the whole broth and / or supernatant has a purity of at least 80 % measured on the total amount of saccharide and its precursor(s) produced by said cell in the whole broth and / or supernatant, respectively.

12. Method according to any one of claims 4 to 11, said method further comprising a step of separating, preferably purifying, said saccharide from the cultivation or incubation.

13. Use of a method according to any one of claims 4 to 12 for the production of a saccharide.

14. Use of a method according to claim 13, wherein said saccharide is selected from the list consisting of or consisting essentially of monosaccharide; phosphorylated monosaccharide; activated monosaccharide; disaccharide; oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); sialylated milk oligosaccharide; neutral (non-charged) milk oligosaccharide; fucosylated milk oligosaccharide; non-fucosylated neutral (noncharged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide;neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non- fucosylated neutral (non-charged) human milk oligosaccharide; O-antigen; enterobacterial common antigen (ECA); the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; an antigen of the human ABO blood group system; an animal oligosaccharide; animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide; plant oligosaccharide selected from the list consisting of N-glycans and O-glycans; fucosylated oligosaccharide; fucosylated oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N- fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N- neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose; sialylated oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N- hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N- neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N- neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N-acetylglucosamine containing neutral (non-charged) saccharide; N-acetylglucosamine containing neutral (non-charged) saccharide selected from the list comprising lacto-N-biose (LNB), N-acetyllactosamine (LacNAc), lacto-N-triose II (LN3), lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N- hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N- hexaose, difucosyl-lacto-N-hexaose, difucosyl-lacto-N-neohexaose (LNnDFH II), difucosyl-para-lacto- N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N-fucohexaose and lacto-N-trifucoheptaose; N- acetylglucosamine containing saccharide; N-acetyllactosamine containing saccharide; lacto-N-biose containing saccharide; non-fucosylated neutral (non-charged) saccharide; chitosan; chitosan comprising oligosaccharide; heparosan; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan sulphate; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.

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